The outer membrane proteins of Stenotrophomonas maltophilia are potential vaccine candidates for channel catfish (Ictalurus punctatus)

Fish & Shellfish Immunology 57 (2016) 318e324

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The outer membrane proteins of Stenotrophomonas maltophilia are potential vaccine candidates for channel catfish (Ictalurus punctatus) Xingli Wang a, Lang Peng b, Kaiyu Wang a, c, *, Jun Wang a, Yang He a, Erlong Wang a, Defang Chen d, Ping Ouyang a, Yi Geng a, Xiaoli Huang d a Department of Basic Veterinary, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China b Department of Aquaculture, Sichuan Province Bureau of Aquatic Products, Chenghua District Donghong Road No. 60, Chengdu 610072, China c Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China d Department of Aquaculture, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 March 2016 Received in revised form 22 August 2016 Accepted 25 August 2016 Available online 26 August 2016

Channel catfish (Ictalurus punctatus) is an important agricultural fish that has been plagued by Stenotrophomonas maltophilia (S. maltophilia) infections in recent years, some of them severe. The outer membrane proteins (OMPs) of S. maltophilia are one of the most immunogenic and highly conserved candidates for vaccine development in aquaculture. The present study investigated OMPs of S. maltophilia as vaccine on immune response and disease resistance against S. maltophilia of channel catfish and investigated the enhancement effect of natural adjuvants Propolis (Pro), FIG polysaccharide (Fcps), Glycyrrhizine (Gly) to OMPs of S. maltophilia for further study. The results indicated that channel catfish injected intraperitoneally with OMPs showed better immune response including leukocytes phagocytosis activity, serum bactericidal activity, complement C3, IgM level and an increasement of resistance against S. maltophilia compared to the control group. Moreover, Pro, Fcps and Gly could enhance the immune response of OMPs. The relative percent of survival (RPS) was 73.33%, 66.67%, 63.33%, 60%, 0% in fish injected OMPs þ Pro, OMPs þ Fcps, OMPs þ Gly, OMPs and 0.65% normal saline, respectively. These results suggested that OMPs used as vaccine could induce and stimulate immune response and enhance disease resistance in channel catfish, especially for Pro as immunoenhancer. Results revealed that OMPs were an effective vaccine against S. maltophilia in channel catfish. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Channel catfish Stenotrophomonas maltophilia Outer membrane proteins Immune effect

1. Introduction The channel catfish (Ictalurus punctatus), one of the most dominant aquaculture species in China, suffer from seriously damage due to a bacterial pathogen Stenotrophomnas maltophilia (S. maltophilia) in recent years [1]. S. maltophilia is a member of the genus Stenotrophomonas, which distributes in the water, soil, plant roots, animals and peoples' body surface and so on [2,3]. It can cause common diseases such as pneumonia, bacteremia and sepsis [4]. After 2000, the bacterium have infected terrestrial, aquatic animals and even rice plants, and it could cause death [5]. When

* Corresponding author. Department of Basic Veterinary, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang District Huimin Road No. 211, Chengdu 611130, China. E-mail address: [email protected] (K. Wang). http://dx.doi.org/10.1016/j.fsi.2016.08.054 1050-4648/© 2016 Elsevier Ltd. All rights reserved.

fish infect S. maltophilia, Geng et al. found that the clinical symptoms appeared enteritis and ascites, especially prolapse in the rectum and intussusception in the lower intestine. Moreover, the signs mentioned above appeared in over 80% diseased fish [1,6,7]. It is necessary to find a simple and effective methods of prevention or treatment of S. maltophilia in channel catfish. Vaccines are green intervention to prevent S. maltophilia infection. Outer membrane proteins (OMPs) located on the cell walls of the specific outer membrane of gram-negative bacteria, which are one of the most important surface antigen of the bacteria [8], and have an important role in the interaction between bacteria with hosts in absorption and entry into the host to subvert hostdefense mechanisms [9]. These OMPs not only can stimulate the cellular immunity and humoral immunity, but also can induce the host to produce protective immunity [10]. In recent years, there has been already more research work about OMPs and got enough attention

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[11]. In order to enhance the immunogenicity of vaccine, adjuvants were always used. The adjuvants that have been studied in association with fish vaccines included oil-based adjuvants, aluminumbased adjuvants and natural adjuvants [12]. Most oil adjuvant and aluminum-based adjuvants have toxicity, potential adverse reaction, and high cost make them unfit to be used as a vaccine adjuvant [13]. Nowadays, natural adjuvants such as Propolis (Pro) [14], Ficuscaricapolysaccharide (Fcps) [15], glycyrrhizin (Gly) [16] and have been reported to enhance the immunity of fish. They are vaccine adjuvant with immunomodulatory, antimicrobial, antiinflammatory, antiviral, antitumor, antioxidant, antiparasitic, and anti-diabetic activities [17]. The aim of this work was to investigate the OMPs of S. maltophilia may been a promising candidate vaccine to prevent S. maltophilia infection. We used SDS-PAGE analysis to investigate biological characteristics of the OMPs in vitro. Channel catfish were vaccinated to evaluate its immune protection in vivo. Subsequently, leukocytes phagocytosis activity, serum bactericidal activity, complement C3, and IgM level were used to assess the effect of OMPs. Moreover, as we still have to make further study of the enhancement effect of natural adjuvants Propolis (Pro), Ficuscaricapolysaccharide (Fcps) and glycyrrhizin (Gly) to OMPs of S. maltophilia. All the experimental results indicated that OMPs are one of the promising candidate vaccines against S. maltophilia in channel catfish.

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by centrifugation at 4000g for 30 min. The bacteria were resuspended with 20 mmol PBS then repetitive freeze-thawing between room temperature and 20  C. The sediment was collected at 13000 g for 30 min and resuspended in 2% (W/V) N-lauroylsarcosine sodium salt at 37  C for 30 min and collected at 13000 g for 30 min. In the end, the sediment were dissolved in 20 mmol/L PBS and all extracted products were stored at 20  C until utilised. 2.4. Biological characteristics of OMPs The content of OMPs was determined by Coomassie brilliant blue kit according to the manual. The quality of OMPs was determined with SDS-PAGE. 2.5. Determination the safety of OMPs to channel catfish Sixty channel catfish were divided into six groups of ten fish each and injected intraperitoneally with 0.2 ml OMPs of S. maltophilia (24 mg/kg, 12 mg/kg, 6 mg/kg, 3 mg/kg, 1.5 mg/kg, respectively), the control group received an identical dose of 0.65% normal saline. The clinical symptoms of challenged fish were observed to record and Karbers method was used to test the LD50 of channel catfish in infected fish in 14 days. LD50 was calculated according to the formula:LD50 ¼ log-1[Xm-i(Sp-0.5)] [27]. Xm is the log value of maximal does. Sp is total mortality.

2. Materials and methods 2.6. The preparation of antigen 2.1. Strains and experimental materials Stenotrophomonas maltophilia(CCF00024) was isolated from channel catfish and stored in our lab. Staphylococcus aureus (ATCC25923) was purchased from National Center for Clinical Laboratories. They were cultured in LuriaeBertani broth medium at 28  C. Coomassie brilliant blue kit was purchased from Nanjing Jiancheng Bioengineering Institute. Ficuscaricapolysaccharide(Fcps) crude products were purchased from corona technology development company in Fuzhou. Rough Propolis(Pro) and Glycyrrhizine(Gly) were purchased from Chinese herbal medicine company. 2.2. Fish Fingerling channel catfish (mean weight 50±5 g) were selected from a farm of channel catfish in Meishan (Sichuan Province, China) and fed daily on commercial feed for a minimum of 7 days in a flow water system at 28  C. They were fed on commercial channel catfish feed daily at 3% fish body weight. Fish were randomly sampled for the examination of bacterial recovery from the blood, liver, spleen, and kidney, and no bacteria could be detected in any of the examined fish. All of the animal experiments were performed in accordance with ethical standards. Fish were usually anaesthetized with tricaine methanesulfonate (Sigma-Aldrich, St. Louis, MO, USA) prior to experimental manipulation to minimize suffering. 2.3. Bacteria culture and OMPs extracted The culture media and extracted method used in this study have been described previously [11,18] with some modification. The 24 h S. maltophilia culture was streaking 100 ml of TSB broth by plating to a TSA plate. Plates were incubated for 24 h at 28  C. Bacteria were harvested by scraping the plate with sterile plastic loops into 10 ml centrifuge tubes containing 20 mmol sterile phosphate buffered saline (PBS), pH 7.2. The bacteria was washed three times with 20 mmol PBS and the supernatant was discarded

Methods the Fcps were obtained through hot water extraction and ethanol precipitation, which content was determined by phenol-vitriolic colorimetry [14,28]. Rough Pro is ground, macerated with four-five(w/v) absolute ethanol(95%) for 3 days, and stirred many times every day. The supernate was obtained by centrifugation at 5000g for 10 min and stored in a dark place at 4  C until used. Gly was finished product. It was suspended in a final concentration of 0.65% sterilized saline and stored at 4  C. The extracted OMPs can be divided into four. One is without the adjuvant, the other three mixd with the Pro, Fcps and Gly extracts respectively. The OMPs concentration was adjusted to 0.75 mg/mL, Fcps and Gly element concentration to 50 mg/mL, Pro final concentration to 5 mg/mL [13e15]. All the adjuvanted vaccines above were stored at 4  C until use (no more than three days). 2.7. Detection of immunogen security Fifty channel catfish were divided into five groups of 10 fish each and injected intraperitoneally with 0.2 ml OMPs þ Pro, OMPs þ Fcps, OMPs þ Gly, OMPs and normal saline, respectively. A period of 7 days to continuously observe. 2.8. Fish immunization and challenge 300 channel catfish were divided into five groups of 60 fish each: four vaccine-treated groups and one control group (Each group contains a parallel of 30 fish, total 60). The individual fish in the four vaccine groups, namely, OMPs þ Pro, OMPs þ Fcps, OMPs þ Glys, OMPs, were injected intraperitoneally with 0.2 ml OMPs þ Pro, OMPs þ Fcps, OMPs þ Gly, OMPs, respectively. The control group received an identical dose of 0.65% normal saline. 4 weeks post-vaccination, the fish in four vaccine groups were boosted with OMPs þ Pro, OMPs þ Fcps, OMPs þ Gly, OMPs and normal saline as mentioned above. On the 28th day after booster vaccination, 30 fish were randomly selected from each treatment group, and challenged with

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S. maltophilia in 1  108 cfu/ml by intraperitoneal injection [19]. A period of 14 days after the challenge, the mortality was recorded daily. RPS was calculated according to the formula: RPS ¼ (1[Mortality of vaccinated fish/Mortality of unvaccinated control fish])  100.

respectively.

2.9. Blood collection and separation of leukocytes

According the value of logMr (Mr is relative molecular mass of standard protein, 97.2 kDa、66.4 kDa、44.3 kDa、29.0 kDa、 20.1 kDa and 14.3 kDa), the ingredient of standard protein and the value of Rf (Rf is relative migration) to draw standard curve (Fig. 1). A Coomassie stained sodium dodecyl sulfateepolyacrylamide electrophoresed gel is shown in Fig. 2. The value of Mr can be obtained by the standard curve. The OMPs contain more than 6 proteins from 78.3 to 14.2 kDa with dominant (heavily stained) proteins at 14.2 kDa, 15.9 kDa, 34.5 kDa, 61.5 kDa, and nondominant at 31.8 kDa, 78.3 kDa.

Three fish were taken randomly from each group to collect the blood samples (1.0 ml) from the caudal vein at 2w, 4w, 6w and 8w post-vaccination, respectively. Individual fish was sampled only once to avoid multiple bleeding and handling stress on the fish. 0.5 mL of blood samples was centrifuged at 4000 g for 10 min at 4  C to collect serum which was stored at 20  C until use. The other 0.5 ml of the blood sample with heparin sodium was used for the separation of lecukocytes according to the density et al. [29]. gradient centrifugation method described by Ardo Briefly, 1.0 mL histopaque-1119 (Sigma) containing 100 ml bacto haemagglutination buffer (pH ¼ 7.3, Difco, USA) was dispensed into siliconised tubes. 1.0 ml of a mixture of histopaque-1077 and haemagglutination buffer and 0.5 ml blood sample was layered carefully on the top of the gradient. Sample preparations were centrifuged at 700 g for 20 min at 4  C. After centrifugation, plasma was collected and stored at 80  C for future analysis. Separated leukocytes were gently removed and dispensed into siliconised tubes, containing phenol red free Hank's Balanced Salt Solution (HBSS). Cells were washed twice in HBSS and adjusted to 2  106 viable cells/ml. 2.10. Determination of immune index 2.10.1. Detection of the leukocyte phagocytic activity Phagocytosis activity of blood leukocytes was determined spectrophotometrically by the method of Seeley et al. [20]. 2.10.2. Detection of the phagocyte bactericidal activity The bactericidal capacity of phagocyte was determined using the Thiazolyl Blue Tetrazolium Blue (MTT) assay, which was by the method of Jeffries et al. [21]. The final data were adjusted to give a killing index (KI) by taking the ratio: KI ¼ MTT reduction at time x/ MTT reduction at time 0. A comparison of the MTT bactericidal assay was made with the more conventional assay based on counting bacterial colonies. After killing had been stopped by lysing the phagocyte, the contents of quadruplicate wells were diluted in TSB and spread over agar plates. The number of colonies present were counted 24 h later and expressed as a killing index as for the MTT assay.

3. Results 3.1. Biological characteristics of OMPs

3.2. The immunogen security to channel catfish When five doses (24 mg/kg, 12 mg/kg, 6 mg/kg, 3 mg/kg, 1.5 mg/ kg per fish) of S. maltophilia OMPs were injected to channel catfish, the 14-day cumulative mortalities were 100%, 60%, 20%, 0%, 0%, respectively(Table 1). The concentration of 24 mg/kg and 12 mg/kg made fish appear that they reduced movement, or stayed silent in the corner. In the end, all fish of the two groups were dead at 24 h post-challenge. The 6 mg/kg group has a similar symptoms, but this group was dead in 24e48 h. At the beginning of the 3 mg/kg group and 1.5 mg/kg group showed that fish were swimming slowly or staying silent in the corner, but they returned to normal soon. The control group always showed normal. The symptom to the dead fish included chest and dorsal fin base were severe congestion, anus redness eversion, ascitic fluid, intestinal wall hyperemia, hemorrhages with a mild inflammation, a yellow mucus in the intestines, hepatomegaly. After 14 d, the body surface and viscera were normal when dissected survived fish in low does groups. Finally, according to the Karber method, LD50 was calculated as 9.6 mg/kg. 3.3. Leukocyte phagocytosis activity The leukocyte phagocytosis activity was assessed by spectrophotometry 2w, 4w, 6w and 8w post-vaccination (Fig. 3A). Compared with control group, the leukocyte phagocytosis activity of the four vaccinated groups were slightly above, while there was no obvious difference in OMPs þ Gly group, OMPs group and control group after vaccination. However, There was a significant

2.10.3. Complement C3 assay The serum complement C3 level was determined by C3 detection-kit purchased from Zhejiang erkn biological technology co, .LTD and the usage refer to manual. 2.10.4. Detection of the IgM titers The IgM titers were determined by IgM detection-kit. The kit was purchased from Shanghai Sun biological technology co. LTD and the usage refer to manual. 2.11. Statistical analysis All data were analyzed by one-way analysis of variance and Duncan's test using the SPSS 19.0 package (SPSS Inc., Chicago, IL, USA). All results were shown as mean ± SD, and the differences were considered statistically non-significant, significant and extremely significantly when p > 0.05, p < 0.05 and P < 0.01

Fig. 1. Standard curve of relative molecular weight.

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during 8 weeks of injecting with vaccines and control normal saline. The four vaccinated groups have higher IgM when compared with control group. OMPs group is higher than control group with extremely significantly (P < 0.01) at 2w, 4w and 6w. After booster immunization, OMPs þ Pro group was significantly (P < 0.05) or extremely significantly (P < 0.01) higher than other vaccines groups at 6w and 8w. Moreover, the serum IgM levels of fish in vaccinated groups reached highest at weeks 6. 3.7. Disease resistance The cumulative mortality of channel catfish injected with four vaccines and 0.65% normal saline during 15 days post challenge with S. maltophilia shown in Fig. 4. Monitoring the fish mortality for post challenge revealed that mortality was remarkably lower in the OMPs þ Pro group, OMPs þ Fcps group, OMPs þ Gly group, OMPs group compared to the control group. The relative percent of survival (RPS) was 73.33%, 66.67%, 63.33%, 60%, 0% in fish injected OMPs þ Pro, OMPs þ Fcps, OMPs þ Gly, OMPs and 0.65% normal saline, respectively. 4. Discussion Fig. 2. SDS-PAGE analysis of OMPs from S. maltophilia.

OMPs are one of the important surface antigen of the gram-

Table 1 The mortality of channel catfish injected intraperitoneally with S. maltophilia OMPs. Group

Number

OMPs concentration(mg/kg)

Inject volume(ml)

Death number

Mortality (%)

1 2 3 4 5 6

10 10 10 10 10 10

24 12 6 3 1.5 0

0.2 0.2 0.2 0.2 0.2 0.2

10 6 2 0 0 0

100 60 20 0 0 0

difference in OMPs þ Pro group, OMPs þ Fcps group and control group at the 6w (P < 0.05). 3.4. Phagocyte bactericidal activity The four experimental groups had stronger bactericidal activity than control group in primary immunization and booster immunization, especially the OMPs þ Pro group (Fig. 3B). And the bactericidal activity of all the experimental groups were extremely significantly (P < 0.01) higher than control group at 2w and 6w. However, OMPs þ Pro group was extremely significantly (P < 0.01) higher than other experimental groups at 4w, 6w and 8w. 3.5. Serum C3 levels The serum complement C3 titers were assessed by C3 detectionki at 2w, 4w, 6w and 8w post-vaccination(Fig. 3C). The results illustrated that the complement C3 contents in vaccinated groups were significantly (P < 0.01) increased at weeks 2e8 compared to control group. In addition, OMPs þ Pro was significantly (P < 0.01) or extremely significantly (P < 0.01) higher than other group from 4w to 8w. Moreover, there was no significant difference between OMPs þ Fcps group and OMPs þ Gly group (P > 0.05) in the whole time. 3.6. Serum IgM levels From Fig. 3D showed the serum IgM contents of channel catfish

negative bacteria. So far, a large number of studies have shown that OMPs have good immunogenicity [22e24]. Hence, OMPs are potential vaccine candidates. OMPs extracted from S. maltophilia have been shown to be protective antigens [25]. We concluded that OMPs might also be vaccine candidates to further study. In our previous work, we done some experiments to confirm their security. The results showed that S. maltophilia OMPs had a large lethal dose (LD50 9.6 mg/kg). And the fish have no obvious harm when the concentration of the OMPs reached to 3 mg/kg. After 14d of challenge, the body surface and viscera were normal when dissected the survived fish. The results showed that low doses S. maltophilia OMPs may be a safer vaccine. Adjuvants are usually used as immune enhancer in vaccine. So far the types of adjuvants allowed in commercial fish vaccines are limited, however it is documented that these adjuvants, especially oil-based adjuvant (such as Freund's adjuvant), have deleterious side effects [12]. Therefore, adjuvants obtained from natural sources being biodegradable and biocompatible have received attention with regard to fish. In this study, we used Pro, Fcps, Gly as immune booster, which are natural product, and they are safer, less toxic and cheaper. Our result showed that Pro, Fcps, Gly are good immune enhancer, and Pro is best. In fish, non-specific immunity usually precedes the specific immune response, activates and determines the nature of the specific immunity and join in the maintenance of homeostasis [26]. In fish, leukocyte is considered to be an important part of the nonspecific immunity, and it is an important indicator of the body's defence capabilities [27e29]. In this study, although there was no

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Fig. 3. Leukocytes phagocytosis activity (A), serum bactericidal activity (B), complement C3 (C) and serum IgM levels (D) of channel catfish in vaccined groups and control group. Note: Data are presented with the means ± standard deviation. Letter a, b, c, d, e represent difference (P < 0.05) between any one of the groups and OMPs þ Pro group, OMPs þ Fcps group, OMPs þ Gly group, OMPs group and control group, respectively. Data are presented with the means ± standard deviation. Letter A, B, C, D and E represent the significant difference (P < 0.01) between any one of the groups and OMPs þ Pro group, OMPs þ Fcps group, OMPs þ Gly group, OMPs group and control group, respectively.

Fig. 4. The cumulative mortality of channel catfish injected with four vaccines and 0.65% normal saline during 15 days post challenge with S. maltophilia.

obvious difference in OMPs group and control group after vaccination, there was a significant difference in OMPs þ Pro group, OMPs þ Fcps group and control group at the 6w (P < 0.05). It is speculated that Gly and Fcps could strengthen the leukocyte

phagocytic activity of OMPs. Phagocyte bactericidal activity is an important non-special immune indices in fish. The result showed that S. maltophilia OMPs could enhance phagocyte bactericidal activity in channel catfish and Pro could enhance the effect of OMPs.

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The complement system plays an important role in host defense against pathogenic is a major humoral system of non-specific immunity. The function of complement includes production of opsonin molecules, anaphylatoxin, direct killing of pathogens and maintaining homeostasis [30]. Complement component 3 is a key molecule in the complement system and its activation is essential for all the important functions performed by this system. C3 is a central protein of the complement system, this versatile and flexible molecule interacts with various proteins to perform its functions [31,32]. The results showed that C3 is markedly stimulated by OMPs, and there was an extremely significantly (P < 0.01) higher than control groups. Although Pro, Fcps and Gly could strengthen the C3 of OMPs, the best effect is Pro. IgM is an important immunoglobulin class. It is important in phylogenetic research being the first immunoglobulin to appear in evolution and commonly the only immunoglobulin class described in fish. It is also important in an ontogenetic context and the major antibody of primary response in higher vertebrates [33]. Fish IgM is important to the fish farming industry for effective prevention and control of various fish diseases [34]. In this study, in the examing indexs of IgM in serum, OMPs group mostly displayed better than control group. Besides, Pro, Fcps and Gly also could strengthen the IgM of OMPs. RPS is an important parameter to evaluate the effects of vaccines. In the present study, the vaccine OMPs exhibited moderate immunoprotective efficacy, while the relative percent of survival (RPS) was 73.33%, 66.67%, 63.33%, 60%, 0% in fish injected OMPs þ Pro, OMPs þ Fcps, OMPs þ Gly, OMPs and 0.65% normal saline, respectively. Thus, RPS demonstrated that OMPs had a moderate capacity for immune protection., and Fcps, Pro and Gly could enhance the immune protection of OMPs. 5. Conclusions In conclusion, our study demonstrate that, the OMPs of S. maltophilia possesses immunogenicity and immune protection against S. maltophilia infection in channel catfish. As far as the effect on protection efficacy is concerned, OMPs has a good effective avaccine, probably due to its ability to induce a much broader range of immune response. And Fcps, Pro and Gly could enhance the immune protection of OMPs against S. maltophilia infection in channel catfish. The OMPs shows promise as a vaccine candidate in aquaculture. The vaccine candidate is being further evaluated based on their protective efficacy against challenge with both homologous and heterologous strains of S. maltophilia in animal models. The role of this protein in the pathogenesis of S. maltophilia will also be for further study. Conflicts of interest The authors have declared no conflict of interest. Acknowledgments We thank Tao Liu, Yukun Zeng and Qian Yang participated in vaccinating and infecting fish; This work supported by Sichuan Technology Support Plannings (No.2014NZ0003) and (No.2014JY0143), Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0848). References [1] Y. Geng, K. Wang, D. Chen, X. Huang, M. He, Z. Yin, Stenotrophomonas maltophilia, an emerging opportunist pathogen for cultured channel catfish, Ictalurus punctatus, in China, Aquaculture 308 (2010) 132e135.

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