Membrane interaction of chrysophsin-1, a histidine-rich antimicrobial peptide from red sea bream

Chrysophsin-1 is an amphipathic alpha-helical antimicrobial peptide produced in the gill cells of red sea bream. The peptide has broad range activity against both Gram-positive and Gram-negative bacteria but is more hemolytic than other antimicrobial peptides such as magainin. Here we explore the membrane interaction of chrysophsin-1 and determine its toxicity, in vitro, for human lung fibroblasts to obtain a mechanism for its antimicrobial activity and to understand the role of the unusual C-terminal RRRH sequence. At intermediate peptide concentrations, solid-state NMR methods reveal that chrysophsin-1 is aligned parallel to the membrane surface and the lipid acyl chains in mixed model membranes are destabilized, thereby being in agreement with models where permeabilization is an effect of transient membrane disruption. The C-terminal RRRH sequence was shown to have a large effect on the insertion of the peptide into membranes with differing lipid compositions and was found to be crucial for pore formation and toxicity of the peptide to fibroblasts. The combination of biophysical data and cell-based assays suggests likely mechanisms involved in both the antibiotic and toxic activity of chrysophsins.     

Immunohistochemistry,ultrastructure and pathology of gills of Abramis brama from Lake Mondsee,Austria, infected with Ergasilus sieboldi (Copepoda)

Immunohistochemical, ultrastructural and pathological studies were carried out on the gills of bream Abramis brama (L.) from Lake Mondsee, Austria, that were naturally infected with Ergasilus sieboldi Nordmann, 1832. Of a total of 14 specimens of bream examined, the gills of 7 (50%) were parasitized with this copepod and the intensity of infection ranged from 1 to 23 crustaceans per host. Histopathological investigations of infected gill showed extensive tissue damage due to attachment and feeding of the crustacean. Parasites attached close to the base of filaments near the gill arch. Pressure exerted by the ectoparasite attached to the lateral margin of the gill filaments induced atrophy of the secondary lamellae. Tissue reactions included hyperplasia and mucous cell proliferation of the respiratory epithelium. Mucous cells displayed an intense immunohistochemical reactivity with the anti-nitric oxide synthase antibody. In parasitized primary and secondary lamellae, a high number of eosinophilic granular cells and rodlet cells were noticed. Rodlet cells represent an inflammatory cell type closely linked to other piscine inflammatory cells. Presence of a high number of inflammatory cells at the site of E. sieboldi attachment is related to intense host cellular reaction.     

Localization of group IB phospholipase A(2) isoform in the gills of the red sea bream,Pagrus (Chrysophrys) major

We previously reported that PLA(2) activity in the gills is higher than that in other tissues in red sea bream and purified PLA(2) from the gills belongs to the group IB PLA(2) as well as other red sea bream PLA(2)s. In this study, we reconfirmed that the level of PLA(2) activity is extremely high in the gills compared with other tissues, and gill PLA(2) was detected only in the gills by immunoblotting and inhibition test using anti-gill PLA(2) monoclonal antibody. The level of PLA(2) activity and protein expression in the gills are well correlated. Fish can be roughly divided into high and low groups based on the level of PLA(2) activity. Gill PLA(2) was detected in the gills of the high group, but not the low group by immunoblotting. In the gills of the high group, gill PLA(2) was detected in the mucous cells and pavement cells located on the surface of gill epithelia by immunohistochemistry. On the other hand, positive signals were observed only in the mucous cells by in situ hybridization. We also isolated inactive proPLA(2), having AR propeptide, preceding the mature enzyme from the gill extract. These results suggest that gill PLA(2) is synthesized as an inactive proPLA(2) in the mucous cells and is secreted to the surface of gill epithelia.     

Distribution of peptidase activity in teleost and rat tissues

Peptides play important roles in cell regulation and signaling in many tissues. The actions of peptides are regulated by peptidases. Although the activity of these enzymes has been thoroughly characterized in mammals, little is known about their presence or function in fish. In the present study, we compared the activity of several peptidases in selected tissues (pituitary gland, different brain areas, kidney and gills) of the gilthead sea bream and rainbow trout with that found in similar rat tissues (lungs studied in place of gills). Soluble puromycin-sensitive aminopeptidase showed the highest values in the pituitary gland of the sea bream, whereas the membrane-bound form was found to be more active in the trout kidney. Very high levels of activity of aminopeptidase N were detected in trout and sea bream plasma. In contrast, the highest levels of activity of aminopeptidase B were found in rat tissues, with the exception of the gills of the trout. Aminopeptidase N levels tended to be higher in sea bream tissues with respect to those of trout. In contrast, the level of activity of aminopeptidase B was found to be consistently much higher in trout tissues than in those of the sea bream. Prolyl endopeptidase activity was principally detected in the pituitary gland and in the brain areas of teleosts. These differences between species could be related to different mechanisms of osmoregulation in saltwater- and in freshwater-adapted fish.     

Red sea bream iridoviral disease

The first outbreak of red sea bream iridoviral disease caused by red sea bream iridovirus (RSIV) was recorded in cultured red sea bream Pagrus major in Shikoku Island, Japan in 1990. Since 1991, the disease has caused mass mortalities of cultured marine fishes not only red sea bream but also many other species. The affected fish were lethargic and exhibited severe anemia, petechiae of the gills, and enlargement of the spleen. The causative agent was a large, icosahedral, cytoplasmic DNA virus classified as a member of the family Iridoviridae and was designated as red sea bream iridovirus (RSIV). The genome of RSIV is liner dsDNA and considered to be circularly permitted and terminally redundant like other iridoviruses. The length of physical map of RSIV genome is 112,415bp. An indirect immunofluorescence test with a monoclonal antibody and PCR are commonly used for the rapid diagnosis of RSIV infected fish in the field. For the control of this disease, a formalin-killed vaccine against red sea bream iridoviral disease was developed and now commercially available.     

A morphological study on posterior gills of the mangrove crab Ucides cordatus

Morphological and histological studies on posterior gills of the mangrove crab Ucides cordatus showed that the 5th gill (of 7) has a larger surface area and a greater number of lamellae compared to the 6th gill. Regular separation of gill lamellae, important when the gill is in air, is maintained by enlargements of the marginal canals. Conical, spine-like structures along the efferent vessel of both 5th and 6th gills were also observed. In addition, pillar cells, a discontinuous lamellar septum and a hypobranchial artery were observed. The presence of valve-like structures near the efferent vessel was also indicated. These structures, together with the pillar cells, may have a role in directing the hemolymph flow towards certain gills during particular physiological states. Localization of osmoregulatory epithelia in the lamellae of both gills was inferred from dimethylaminostyrylethylpyridiniumiodine staining. Apparently gills 5 and 6 have osmoregulatory epithelial cell patches of similar area, corresponding to 43% and 38% of the total lamellae area, respectively. However, their localization is quite different. Gill number 5 osmoregulatory patches seem to be restricted to the afferent region of the lamella whereas in gill number 6, they are more dispersed over the entire lamella. These differences may be related to the particular functional characteristics of these gills.     

Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream

Immunocytochemical, light microscopy and ultrastructural studies were conducted on gill of sea bream, Sparus aurata L., naturally parasitized with the important parasitic copepod Ergasilus sp. to assess pathology and cellular responses. Thirty-seven S. aurata were examined from a fish farm; 26 (70%) were parasitized, with infection intensity ranging from 3 to 55 parasites per fish. Hosts were divided into two groups, lightly infected fish (<15 parasites per fish) and heavily infected fish (>15 parasites per fish). In histological sections, the copepod encircled gill lamellae with its second antennae, compressed the epithelium, provoked hyperplasia and hemorrhage, occluded arteries and often caused lamellar disruption. Fusion of the secondary lamellae due to epithelial hyperplasia was common in all infected fish; heavily infected fish showed more intense branchial inflammation. In both healthy and infected fish, mast cells (MCs) were free within the connective tissue inside and outside the blood vessels of the primary lamellae and made close contact with vascular endothelial cells, mucous cells and rodlet cells (RCs). MCs were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide (AMP) piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of MCs that were positive. These MCs were more abundant in gills of heavily infected fish than in either lightly infected or uninfected fish (ANOVA, P<0.05). Our report documents the response of gill to ectoparasite infection and provides further evidence that mast cells and their AMPs may play a role in responding to branchial ectoparasite infections.     

Molecular characterization of peroxisome proliferator-activated receptors (PPARs) and their gene expression in the differentiating adipocytes of red sea bream Pagrus major

To investigate the molecular mechanism of fish adipocyte differentiation, the three subtypes of PPAR genes (alpha, beta and gamma) were characterized in a marine teleost red sea bream (Pagrus major). The primary structures of red sea bream PPARs exhibited high degrees of similarities to their mammalian counterparts, and their gene expression was detected in various tissues including adipose tissue, heart and hepatopancreas. During the differentiation of primary cultured red sea bream adipocytes, three PPARs showed distinct expression patterns: The alpha subtype showed a transient increase and the beta gene expression tended to increase during adipocyte differentiation whereas the gene expression level of PPARgamma did not change. These results suggest that they play distinct roles in adipocyte differentiation in red sea bream. In the differentiating red sea bream adipocytes, mammalian PPAR agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2), ciglitazone and fenofibrate did not show clear effects on the adipogenic gene expression. However, 2-bromopalmitate increased the PPARgamma and related adipogenic gene expression levels, suggesting the gamma subtype plays a central role in red sea bream adipocyte differentiation and in addition, fatty acid metabolites can be used as modulators of adipocyte function. Thus our study highlighted the roles of PPARs in fish adipocyte differentiation and provided information on the molecular mechanisms of fish adipocyte development.     

Immunohistological classification of ionocytes in the external gills of larval Japanese black salamander,Hynobius nigrescens Stejneger

In this cytological and immunohistological study, we clarified the localization of the membrane transporters Na⁺, K⁺‐ATPase (NKA), vacuolar‐type H⁺‐ATPase (VHA), and epithelial sodium channel (ENaC) and distinguished ionocyte subtypes in the gill of the Japanese salamander (Hynobius nigrescens). In larvae (IY stages 43–65), NKA immunoreactivity was observed on the basolateral plasma membrane in more than 60% cells and less than 20% cells in the primary filaments and secondary lamellae of the external gills, respectively. VHA immunoreactivity was observed on the apical membrane of some epithelial cells in the secondary lamellae of the external gills. High ENaCα immunoreactivity was widely observed on the apical cell membrane of a population of squamous cells, presumably pavement cells (PVCs), and mitochondria‐rich cells (MRCs), in the primary filaments and secondary lamellae of the external gills. Using double immunofluorescence microscopy, epithelial cell types involved in ionic regulation were characterized and divided into three ionocyte types: NKA‐, NKA‐ and ENaC‐, and VHA‐positive cells. VHA‐immunoreactive cells as well as NKA‐positive cells were observed during IY stages 43–65 of the salamander larvae. During late stages of metamorphosis, NKA, VHA, and ENaCα immunoreactivities in the external gills decreased and finally disappeared during the completion of metamorphosis (IY stage 68). PVCs and MRCs in the external gills are probably involved in acid–base balance regulation and osmoregulation in urodele amphibian larvae. The results are discussed in relation to the ionocytes previously reported in fish gills and the frog skin epithelium. J. Morphol., 2011. © 2011Wiley‐Liss, Inc.     

A scanning electron microscope study of the gills of the air-breathing catfish, Clarias batrachus L.

Using the scanning electron microscope, the gills of the air-breathing catfish, Clarias batrachus , have been studied. The overall morphology of the gills are similar to other teleosts. In contrast to water-breathing species, however, microridges are absent from the surfaces of the secondary lamellae and only short microvilli are present. Long, convoluted microridges are present on the epithelial cells of the gill filaments. The possible roles of these structures in relation to water flow are discussed.     

Myostatin precursor is present in several tissues in teleost fish: a comparative immunolocalization study

In this study, the distribution of myostatin was investigated during larval and postlarval developmental stages of Sparus aurata(sea bream), Solea solea(sole) and Brachydanio rerio(zebrafish) by immunohistochemistry using antisera raised against a synthetic peptide located within the precursor region of sea bream myostatin. All the three species examined showed the strongest immunoreactivity in red skeletal muscle in juveniles and adults. During larval development of sea bream, strong staining was detected in skin and brain. Immunoreactivity was also found in muscle, pharynx, gills, pancreas and liver. From metamorphosis, immunoreactivity was identifiable in the oesophagus, in the apical portion of the stomach epithelium, in the intestinal epithelium and in renal tubules. In larval zebrafish at hatching, the most intense myostatin immunoreactivity was evident in the skin epithelium. Immunoreactivity was also found in the retina and brain. In the adult, an intense immunostaining occurred in the gastrointestinal tract as well as in the ovary. In sole larvae, immunoreactivity was found in liver and intestine. Our results support the hypothesis suggested earlier that myostatins in fish have retained a different partition (compared with mammals) of the expression patterns and functions which characterized the ancestral gene before the duplication event that gave rise to growth differentiation factor-11 (GDF-11) and GDF-8 (myostatin).     

Fine structure of the respiratory organs of the Climbing perch, Anabas testudineus (Pisces: Anabantidae)

An electron microscopic study has been made of the three respiratory organs of climbing perch. The gill structure is similar to that of the other telcosts but the thickness of the water/blood barrier is much greater, being as great as 20 μm in some specimens. The increased thickness is due to a multilayered epithelium which is thinner (3.5–7 μm) over the marginal channel of the secondary lamellae. The other two main layers, basement membrane and pillar cell flange, are relatively thin (about 1 μm).
The pillar cells have a typical structure, but in certain regions they are contiguous with one another and line well-defined blood channels. Some of the columns of basement membrane material in such regions may be common to adjacent pillar cells.
The air-breathing organs are (a) the lining of the suprabranchial chambers , and (b) the labyrinthine plates attached to the dorsal region of branchial arches. Electron microscopy showed that their structure is well adapted for gas exchange, the air/blood barriers being only 0.12–0.3 μm, comprising an epithelial layer, basement membrane, and thin capillary endothelium. The many parallel blood channels of the respiratory islets of both organs are separated by pillar-like structures which differ from the pillar cells of the secondary lamellae. Thus the hypothesis that the air-breathing organs represent modified gills is not supported by this study.
The fine structure of the non-respiratory region of the air-breathing organs is similar to that of the skin, and includes chemoreceptor-like cells. Evidence concerning the possible homology of pillar cells with plain muscle cells is discussed.     

Characteristics of the antitumor activities in tumor cells and modulation of the inflammatory response in RAW264.7 cells of a novel antimicrobial peptide, chrysophsin-1, from the red sea bream (Chrysophrys major)

The antimicrobial peptide, chrysophsin-1, exhibits antimicrobial activities with similar efficiencies for both gram-negative and gram-positive bacteria. In this study, we examined the antitumor activity and modulation of the inflammatory response of a synthetic chrysophsin-1 peptide. In vitro results showed that chrysophsin-1 had greater inhibitory effects against human fibrosarcoma (HT-1080), histiocytic lymphoma (U937), and epithelial carcinoma (HeLa) cells. LDH release by HeLa cells was comparable to that of an MTS assay after treatment with 1.5-3 μg/ml chrysophsin-1 for 24 h. Under SEM and TEM observations, we found no intact cell membranes after chrysophsin-1 treatment of HeLa cells for 8 h. The suggested mechanism of the cytotoxic activity of chrysophsin-1 was disruption of cancer cell membranes. In addition, we also examined caspase-3, -8, and -9 activities by Western blotting; the results excluded the participation of apoptosis in chrysophsin-1's effect on HeLa cells. Stimulation by lipopolysaccharide induced tumor necrosis factor (TNF)-α which was able to modulate chrysophsin-1 treatment of RAW264.7 cells and inhibited endogenous TNF-α release but did not block its secretion. With data from this study, we demonstrate that chrysophsin-1 has antimicrobial and antitumor activities and modulates the inflammatory response in RAW264.7 cells.     

Ultrastructural study of the early development and localization of Loma salmonae in the gills of experimentally infected rainbow trout

The early ultrastructural stages of Loma salmonae were studied in the gills of experimentally infected rainbow trout. No parasitic stages were identified during the first 2 wk of the infection. By week 3 postexposure (PE), uninucleate and binucleate meronts were recognized within host cells (no xenomas) associated with the capillary channels of secondary lamellae and lamellar arteries. An inflammatory reaction was absent. In secondary lamellae, infected cells were isolated from the capillary lumen, and some were recognized as pillar cells. In lamellar arteries, infected cells were localized beneath the endothelium and not in the lumen. Inflammatory reaction and destruction of parasites inside blood cells in the lumen of secondary lamellae were observed by week 4 PE. Three hypotheses, i.e., isolation, internalization, and evasion, are proposed to explain the localization of the infected cells in the gills. It is concluded that meronts are the earliest parasitic stage observed by week 3 PE, pillar cells are secondarily infected by phagocytosis of infected cells in the blood, endothelial cells of gills are not infected, and inflammatory response to the parasite starts by week 4 PE.     

High toxicity of the novel bloom-forming species Chattonella ovata (Raphidophyceae) to cultured fish

A toxicological study of an axenic cell line of novel species Chattonella ovata Y. Hara et Chihara (Raphidophyceae) revealed that cultured species of sea bream (Pagrus major), horse mackerel (Trachurus japonicus), and yellowtail (Seriola quinqueradiata) were killed by 4.1–6.8 × 10³, 5.4 × 10³, and 2.8 × 10³ cells/mL, respectively. The sensitivity of the gill lamellae to C. ovata differed among the fish species tested. This finding revealed that C. ovata was highly toxic to the cultured fish. Histological examination showed that edema and hyperplasia of the secondary gill lamellae of red sea bream and horse mackerel occurred when exposed to, or killed by C. ovata, whereas severe damage in the gill lamellae was not observed in yellowtail. Chattonella produced high amounts of superoxide anion radicals and hydrogen peroxide, possibly responsible for the fish death observed. Based on the results of this study and occurrence of a red tide by this organism in China in 2001, we consider this organism to be one of the harmful algae in coastal waters. This is the first report demonstrating that C. ovata is highly toxic to fish, and that it produces superoxide and hydrogen peroxide.     

Effect of a novel antimicrobial peptide chrysophsin-1 on oral pathogens and Streptococcus mutans biofilms

Dental caries and pulpal diseases are common oral bacterial infectious diseases. Controlling and reducing the causative pathogens, such as Streptococcus mutans and Enterococcus faecalis, is a key step toward prevention and treatment of the two diseases. Chrysophsin-1 is a cationic antimicrobial peptide having broad-spectrum bactericidal activity against both Gram-positive and Gram-negative bacteria. In this study, we investigated the antibacterial activity of chrysophsin-1 against several oral pathogens and S. mutans biofilms and performed a preliminary study of the antimicrobial mechanism. Cytotoxic activity of chrysophsin-1 against human gingival fibroblasts (HGFs) was investigated. Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and time-kill assay were used to evaluate the killing effect of chrysophsin-1. Scanning electron microscopy (SEM) was used to analyze morphological and membrane change in oral pathogens. Live/Dead staining, in conjunction with confocal scanning laser microscopy (CSLM), was used to observe and analyze S. mutans biofilms. MIC and MBC results demonstrated that chrysophsin-1 had different antimicrobial activities against the tested oral microbes. Lysis and pore formation of the cytomembrane were observed following treatment of the bacteria with chrysophsin-1 for 4h or 24h by SEM. Furthermore, CLSM images showed that chrysophsin-1 remarkably reduced the viability of cells within biofilms and had a significantly lethal effect against S. mutans biofilms. Toxicity studies showed that chrysophsin-1 at concentration between 8 μg/ml and 32 μg/ml had little effect on viability of HGFs in 5 min. Our findings suggest that chrysophsin-1 may have potential clinical applications in the prevention and treatment of dental caries and pulpal diseases.     

Effects of long-term exposure to different salinities on the location and activity of Na+-K+-ATPase in the gills of juvenile mitten crab, Eriocheir sinensis

The euryhalinity of mitten crab, Eriocheir sinensis, is based on osmoregulation, and thus on the activity of Na(+)-K(+)-ATPase. We studied location and activity of this enzyme in gills of juvenile crabs exposed to 5 per thousand, 25 per thousand, and 40 per thousand salinity. The posterior gills showed always a high number of immunopositive cells (IPC), staining with fluorescent antibody against Na(+)-K(+)-ATPase, covering at 5 per thousand the entire lamellae. At 25 per thousand, they showed fewer IPC which occurred only at the bases of the lamellae. Enzyme activity was consistently higher in posterior than in anterior gills. Low salinity stimulated the activity only in posterior gills. Both histochemical and enzymatic results are consistent with previous ultrastructural observations showing that the epithelial cells of the posterior, but not the anterior gills exhibit typical traits of ionocytes. While an increase in Na(+)-K(+)-ATPase activity at a reduced salinity is consistent with a strong hyper-osmoregulatory capacity in juvenile crabs, a low activity at an enhanced salinity suggests a physiological response, directed towards a reduction of Na(+) uptake. The activity increase of ion-transporting enzymes is directly related to spatial changes in their distribution along the osmoregulatory tissue, i.e. an enhanced number of IPC scattered along the entire lamellae. In juveniles, this allows for successful development and growth at reduced salinities.     

Genotoxic and Histopathological Effects of Water Pollution on Two Fish Species, <Emphasis Type="Italic">Barbus capito pectoralis</Emphasis> and <Emphasis Type="Italic">Chondrostoma nasus</Emphasis> in the Büyük Menderes River,Turkey

The genotoxic and histopathological effects of water pollution were investigated on two fish species caught from the Buyuk Menderes River and from its tributary, the Cine Stream. The Buyuk Menderes basin is an important agricultural area in Turkey. The levels of copper, zinc, cadmium, cobalt, and lead were measured at the surface of the water and in gills, liver, and muscle tissue of Chondrostoma nasus and Barbus capito pectoralis. In some tissues, the concentrations of some of these metals exceeded acceptable levels for human consumption. Zinc was found to be the most abundant metal in water and tissues. Maximal metal accumulation was observed in the liver. To detect the genotoxic potential of contaminants, the formation of micronucleus in erythrocytes was used as indicator of chromosomal damage. The frequency of micronucleus formation did not show significant differences between locations and controls in B. capito pectoralis caught from three locations and C. nasus from two locations. The histological changes included significant decreases of the mean lengths of primary and secondary lamellae. In gills epithelia, we observed cellular proliferation that developed Because of secondary lamellae fusion, ballooning degenerations, or club deformation of secondary lamellae and cystic structures in secondary lamellae. In the liver, the changes included swollen and ruptured parenchymal cells, loss of cord structure, vacuoles filled with cellular debris, focal necrosis, and a significant increase in Kupffer cells.     

Histomorphological alterations in organs of the plaicePleuronectes obscurus from a polluted part of Peter the Great Bay, Sea of Japan

This paper describes the results of a histological study of the internal organs of the plaicePleuronectes obscurus from a polluted part of Amurskii Bay, Sea of Japan. A variety of histomorphological alterations were found in the gills and liver, while the kidney and spleen were affected to a lesser degree. The most frequent lesions in gills were edemas of gill lamellae, epithelial detachments, fusions of the secondary lamellae, hypertrophies and hyperplasias of respiratory cells, pigment accumulations, the presence of parasites, increased quantities of mucous cells, lymphocyte infiltrations, and disturbances of blood circulation. Characteristic morphological changes in the liver were lipid and hydropic dystrophy, pigment accumulation, and the presence of regenerative and necrotic foci. Telangiectasia, globate filaments, and xenomas were found in the gills ofP. obscurus for the first time. These morphological alterations appear to be a result of the chronic effect of pollution in Amurskii Bay.     

海水养殖真鲷最小弧菌外毒素的免疫学特性初步研究

按常规方法免疫健康家兔进行外毒素抗体的制备。将制备的兔抗血清与等体积的外毒素混合,37℃下作用1h后,观察毒素的溶血性及细胞毒性,取免疫前家兔血清(零号血清)作阴性对照,用毒素作阳性对照。结果表明,兔抗毒素血清能中和毒素,抑制毒素对人血细胞的溶血性及对Vero细胞的细胞毒性,中和毒素的兔抗毒素血清最高稀释度为1∶256,毒素对照显示出溶血性及细胞毒性。毒素与一定浓度的嗜水气单胞菌HEC毒素抗血清、霍乱肠毒素抗血清在37℃下分别作用1 h后,观察其溶血性及细胞毒性。结果表明,这2种抗血清都不能中和毒素,毒素与这2种抗血清作用后,仍然保持其溶血性及细胞毒性。用浓度为0.2%的福尔马林对浓度为5mg/mL的外毒素进行灭活处理,得到的灭活疫苗为毒素苗。一定稀释度的毒素苗,在初次免疫真鲷2周后利用病原菌对免疫组及对照组的实验鱼进行人工攻毒感染,毒素苗的免疫保护率可达80%,强化免疫后,免疫保护率有所提高。注射免疫的免疫保护率比浸泡免疫的高。