Vaccination trials of sea bass,Dicentrarchus labrax (L.), against Photobacterium damsela subsp. piscicida,using novel vaccine mixtures

Bacterial cells of the marine fish pathogen Photobacterium damsela subsp. piscicida were grown in novel culture media. A mixture of whole cells and extracellular components was inactivated and used in bath, intraperitoneal (i.p.) and oral vaccination of sea bass, Dicentrarchus labrax, employing two sizes of fish. A commercial vaccine was used for comparative purposes. Control and immunized fish were either bath or intraperitoneally challenged 6 and 12 weeks post-vaccination. Small fish had significantly higher relative percentage survival with the novel vaccine mixture both at 6 and 12 weeks post-vaccination by bath, in comparison with the commercial vaccine. No protection was afforded at 6 or 12 weeks post-immunization by either vaccine after challenge via i.p. injection. Sea bass (1.5-2 g) intraperitoneally vaccinated with various adjuvanted vaccine mixtures were not protected against pasteurellosis. In contrast, larger sea bass (20 g) benefited from vaccination with the novel vaccine mixtures. Intraperitoneal challenge with the pathogen resulted in protection in both fish groups vaccinated with novel vaccine mixtures, whereas control fish suffered high mortalities (> 80%). Orally vaccinated fish were immersion challenged with the pathogen. At 6 and 12 weeks post-vaccination the control fish had a high mortality and the fish vaccinated with the novel vaccine mixture achieved good protection.     

Effects of oral vaccination against vibriosis in turbot, Scophthalmus maximus (L.), and sea bass, Dicentrarchus labrax (L.)

Abstract. Turbot, Scophthalmus maximus (L.), and sea bass, Dicentrarchus labrax (L.), are susceptible to vibriosis, a septicaemia caused in France by Vibrio anguillarum 408. A bacterin produced with this strain by Rhône–Merieux has been tested orally in both species and, as a comparison, by intraperitoneal injection. During a challenge carried out 4 weeks later by intraperitoneal inoculation, the protection observed in orally vaccinated fishes was significant (72 % in sea bass and 70 % in turbot). Eleven weeks after vaccination, this protection is still significant. The mean titre of serum agglutinins was weakly increased in orally vaccinated fishes. Bacteriostatic antibodies have also been searched for in serum, but compared to agglutination, the applied technique leads to lower titres and it has not been possible to prove the presence of such antibodies after oral vaccination. Passive immunizations using serum from orally vaccinated fishes (2 months after vaccination) confers some protection against a challenge carried out by inoculation of virulent vibrio. Therefore, the production of serum factors following oral vaccination of sea bass and turbot may be involved in generalized protection of fish.     

Characterization of serum and mucosal antibody responses and relative per cent survival in rainbow trout, Oncorhynchus mykiss (Walbaum), following immunization and challenge with Flavobacterium psychrophilum

Serum and mucosal antibody responses of juvenile rainbow trout, Oncorhynchus mykiss, were characterized by enzyme‐linked immunosorbent assay (ELISA) following immunization with various preparations of formalin‐killed Flavobacterium psychrophilum cells. The protective nature of these preparations was then determined by immunizing rainbow trout fry and challenging with the bacterium. Juvenile rainbow trout immunized intraperitoneally (i.p.) with formalin‐killed F. psychrophilum emulsified with Freund's complete adjuvant (FCA), and i.p. with formalin‐killed F. psychrophilum either with or without culture supernatant generated significant serum antibody responses by 6 and 9 weeks, respectively. Significant mucosal antibody responses were detected by 9 weeks only in fish immunized i.p. with killed F. psychrophilum/FCA. Following immunization and bacterial challenge of rainbow trout fry, protective immunity was conferred in F. psychrophilum/FCA and saline/FCA groups with relative per cent survival values of up to 83 and 51, respectively. Significant protection was not observed in treatment groups immunized by immersion or i.p. without adjuvant at the challenge doses tested. Results suggest that stimulation of non‐specific immune factors enhances the ability of fish to mount a protective immune response, but specific antibody appears necessary to provide near complete protection. In this study, an ELISA was developed to monitor anti‐F. psychrophilum antibody production in trout. The relationship of such responses to protective immunity suggests that future vaccination strategies against coldwater disease may require stimulation of both the innate and adaptive arms of the immune response.     

Oral immunization of rainbow trout, Salmo gairdneri Richardson, against vibriosis with vaccines protected against digestive degradation

Abstract. In order to reduce digestive degradation of vaccines against vibriosis when administered orally to fish, two methods of protecting the antigens were tested. Lyophilized vaccine was either incorporated into a slow-release pellet or coated by an acid-resistant film. These vaccines were compared to both oral vaccination by an unprotected vaccine as well as to standard vaccination methods of immersion and injection. However, mortalities after an artificial challenge showed that the efficacy of the protected vaccines was lower than that of the unprotected vaccine given orally and the standard vaccination procedures. The reason for this is probably that the important antigens of vibriosis vaccines are lipopolysaccharides which are little affected by gastric digestion, and the coating of the vaccine or incorporation into slow-release pellets resulted only in reduced absorption of the antigens.     

Efficacy of furunculosis vaccines in turbot, Scophthalmus maximus (L.): evaluation of immersion, oral and injection delivery

The commercial furunculosis vaccine Aquavac Furovac 5 and an autogenous vaccine, based on the challenge strain, induced immune protection in turbot, Scophthalmus maximus (L.), as shown in challenge tests 120 days post-immunization by injection (relative percentage of survival, RPS = 72-99%). This protective effect lasted for at least 6 months post-immunization at appreciable levels (RPS = 50-52%). Neither the autogenous vaccine nor the commercial vaccine was able to induce significant levels of protection against Aeromonas salmonicida in turbot when administered by immersion. Antibody levels were high or moderate in fish vaccinated by injection with the different vaccines and very low in fish vaccinated by immersion. The field results show that delivering an oral boost after the primary vaccination by injection did not enhance protection of turbot against furunculosis and that water-based (autogenous vaccine) and oil adjuvanted (Alpha Ject 1200) vaccines administered by injection conferred similar levels of protection (RPS > 80%) in turbot.     

Immune protection in carp, Cyprinus carpio L., after immunization with Aeromonas hydrophila crude lipopolysaccharide

Abstract. Vaccination with crude lipopolysaccharide (LPS) induced better protection against infection with Aeromonas hydrophila in carp than vaccination with formalin killed vaccine. Dipping fish in vaccine for 2 h at 25°C was more effective than intraperitoneal injection of the vaccine in procedural simplicity, lower stress loading and the degree of protection acquired. In carp immunized with crude LPS by the dip method, antibodies were not detected by bacteriai agglutination, passive haemagglutination and the agar diffusion tests. The results indicate that the protection against A. hydrophila infection in carp is not dependent on humoral immunity.     

罗非鱼海豚链球菌疫苗及其免疫效果的研究

用筛选到的海豚链球菌(Streptococcus iniae)临床分离菌株CMS005,甲醛灭活制备成疫苗并对其进行了注射、浸泡和口服免疫奥尼罗非鱼的效果研究。结果显示:实验室水族箱试验的注射、浸泡和口服免疫的最佳免疫保护率分别为90.5%、61.9%和14.3%,水泥池小网箱试验的最佳免疫保护率分别为100%、86.1%和66.7%。免疫剂量对浸泡免疫效果的影响大于注射和口服免疫,加强免疫可显著提高浸泡和口服免疫效果,超声波处理可以提高浸泡免疫效果。注射和口服免疫7 d和15 d后均可控制罗非鱼海豚链球菌病的继续发生;未免疫组罗非鱼月累计死亡率为4%~16%,而免疫组罗非鱼月累计死亡率低于0.5%;疫苗产生的免疫保护力可持续2~3个月。     

Positive correlation between Aeromonas salmonicida vaccine antigen concentration and protection in vaccinated rainbow trout Oncorhynchus mykiss evaluated by a tail fin infection model

Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin‐killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin‐killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710‐1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60‐s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine.     

Oral DNA vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), against infectious haematopoietic necrosis virus using PLGA [Poly(D,L-Lactic-Co-Glycolic Acid)] nanoparticles

A DNA vaccine against infectious haematopoietic necrosis virus (IHNV) is effective at protecting rainbow trout, Oncorhynchus mykiss, against disease, but intramuscular injection is required and makes the vaccine impractical for use in the freshwater rainbow trout farming industry. Poly (D,L-lactic-co-glycolic acid) (PLGA) is a U.S. Food and Drug Administration (FDA) approved polymer that can be used to deliver DNA vaccines. We evaluated the in vivo absorption of PLGA nanoparticles containing coumarin-6 when added to a fish food pellet. We demonstrated that rainbow trout will eat PLGA nanoparticle coated feed and that these nanoparticles can be detected in the epithelial cells of the lower intestine within 96 h after feeding. We also detected low levels of gene expression and anti-IHNV neutralizing antibodies when fish were fed or intubated with PLGA nanoparticles containing IHNV G gene plasmid. A virus challenge evaluation suggested a slight increase in survival at 6 weeks post-vaccination in fish that received a high dose of the oral vaccine, but there was no difference when additional fish were challenged at 10 weeks post-vaccination. The results of this study suggest that it is possible to induce an immune response using an orally delivered DNA vaccine, but the current system needs improvement.     

Efficacy of a polyvalent injectable vaccine against Flavobacterium psychrophilum administered to rainbow trout (Oncorhynchus mykiss L.)

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country‐wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin‐inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin‐killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate‐buffered saline or vaccine formulated with Montanide by intra‐peritoneal (i.p.) injection and challenged by intra‐muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post‐vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un‐adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish.     

Optimization by orthogonal array design and humoral immunity of the bivalent vaccine against Aeromonas hydrophila and Vibrio fluvialis infection in crucian carp (Carassius auratus L.)

Aeromonas hydrophila and Vibrio fluvialis are the causative agents of a serious haemorrhagic septicaemia that affects a wide range of freshwater fish in China. In order to develop a bivalent anti‐A. hydrophila and anti‐V. fluvialis formalin‐killed vaccine to prevent this disease, an orthogonal array design (OAD) method was used to optimize the production conditions, using three factors, each having three levels. The effects of these factors and levels on the relative per cent survival for crucian carp were quantitatively evaluated by analysis of variance. The final optimized formulation was established. The data showed that inactivation temperature had a significant effect on the potency of vaccine, but formalin concentration did not. The bivalent vaccine could elicit a strong humoral response in crucian carp (Carassius auratus L.) against both A. hydrophila and V. fluvialis simultaneously, which peaked at 3 or 5 weeks respectively. Antibody titres remained high until week 12, the end of the experiment, after a single intraperitoneal injection. The verification experiment confirmed that an optimized preparation could provide protection for fish at least against A. hydrophila infection, and did perform better than the non‐optimized vaccine judged by the antibody levels and protection rate, suggesting that OAD is of value in the development of improved vaccine formulations.     

Immune response of sea bass <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> to <Emphasis Type="Italic">Tenacibaculum maritimum</Emphasis> antigens

ABSTRACT:   Seawater fishes are affected by a pathology commonly called 'myxobacteriosis', caused by Tenacibaculum maritimum (formerly Flexibacter maritimus ). The disease is characterized by fin erosion and necrotic ulcers of skin and muscle, and by low but constant mortality in cultured marine fish; in Italy is one of the most important and widely spread diseases affecting sea bass Dicentrarchus labrax , gilthead seabream Sparus aurata , sharp-snouted bream Diplodus puntazzo , white bream Diplodus sargus , and six-tooted bream Dentex dentex . In order to obtain an effective vaccine against the disease, formalin killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) preparations were obtained from the T. maritimum strain SPVId and injected intraperitoneally twice into the sea bass. The fish immune response to the preparations was studied: agglutinating antibody titer and in vitro phagocytosis were determined after the first and second injection in order to evaluate whether the preparations are immunogenic or not and if the booster effect took place. The results show that FKC and LPS preparations increased the antibody titer after the first injection when compared to the control sea bass. Moreover, all the preparations stimulated a secondary (booster) response. In vitro phagocytosis of the total blood was significantly higher for all the preparations when compared to the controls, but the crude LPS immunized sea bass showed the highest activity.     

Microencapsulation of a putative probiotic Enterobacter species,C6‐6, to protect rainbow trout,Oncorhynchus mykiss (Walbaum), against bacterial coldwater disease

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD), which has a major impact on salmonid aquaculture globally. An Enterobacter species, C6‐6, isolated from the gut of rainbow trout, Oncorhynchus mykiss (Walbaum), has been identified as a potential probiotic species providing protection against BCWD. This study examined the effects of alginate microencapsulation on the protective efficacy of C6‐6 against BCWD in vivo when administered to rainbow trout fry orally or by intraperitoneal (IP) injection. Viable C6‐6 bacteria were microencapsulated successfully, and this process (microencapsulation) did not significantly deteriorate its protective properties as compared to the administration of non‐microencapsulated C6‐6 bacteria. Both oral and IP delivery of C6‐6 achieved significantly better protection than control treatments that did not contain C6‐6 bacteria. The highest relative percent survival (RPS) resulted from IP delivery (71.4%) and was significantly greater than the highest oral RPS (38.6%). Successful intestinal colonization was not critical to protective effects of C6‐6. The study showed that C6‐6 administration, with or without encapsulation, was a viable choice for protecting fry from BCWD especially when administered intraperitoneally.     

A Comparison of Immersion, Immersion/Oral Combination and Injection Methods for the Vaccination of Channel Catfish Ictalurus punctatus Against Edwardsiella ictaluri

Abstract.— Four different vaccination regimes, including non-vaccinated controls (C), immersion/oral combination (IO). late immersion (LI), and injection (IJ), were evaluated for antibody production and protection from challenge in channel catfish Ictalurus punctatus that were hatched and raised in recirculating culture systems over a 28-wk period. For IO, fry were vaccinated by immersion on day 12 post-hatch and given an oral booster during week 10 post-hatch. The LI and IJ delivered vaccine was also administered at week 10 post-hatch. No titers were detected in the IO group prior to the oral booster at week 10. Mean titers rose sharply after the week 10 vaccinations in all treatment groups, maintained a high level for about 8 wk, and slowly declined over the next 4 wk. Injected fish had significantly higher titers during the primary response period than either LI or IO fish. There was no statistical difference in titers between LI and IO fish during the primary response. After an oral booster delivered during week 22, titers in LI and IO fish increased significantly at week 25, while titers in IJ fish declined despite the booster delivery. An immersion booster delivered during week 25 resulted in significant increases in titers in all vaccinated groups. Of multiple challenges, the only one not marred by concurrent columnaris infections revealed a low, but significant level of protection in IJ fish at 12 wk post-vaccination.     

鲖爱德华菌口服疫苗对斑点鲖的免疫效果

为评价鲖爱德华菌口服微球疫苗对斑点鲖的免疫效果,实验以天然高分子聚合物海藻酸钠和鲖爱德华菌灭活疫苗为材料,制备鲖爱德华菌口服微球疫苗。将实验动物随机分为鲖爱德华菌微球疫苗组、鲖爱德华菌灭活疫苗组、空微球组和对照组,以拌料口服方式进行免疫,通过检测血清中溶菌酶活力、总超氧化物歧化酶(T-SOD)活力、补体替代途径(ACH50)活性等非特异性免疫指标,抗体效价以及相对免疫保护率评价疫苗免疫效果,采用荧光定量PCR检测口服疫苗对斑点鲖免疫相关基因表达量的影响。结果显示,鲖爱德华菌口服微球疫苗能够较长时间增强斑点鲖非特异性免疫功能;血清凝集效价于第5周达到峰值,为1∶16,免疫后第7周仍可检测到特异性抗体;口服鲖爱德华菌微球疫苗的斑点鲖获得的抗鲖爱德华菌相对免疫保护率为60.7%,远高于灭活疫苗组(14.3%)及空微球组(10.7%);荧光定量分析结果显示,攻毒后48 h相比攻毒前各免疫基因表达量均有上调,鲖爱德华菌微球疫苗对受免鱼肾脏、脾脏中免疫基因的表达影响尤为明显。结果表明,鲖爱德华菌口服微球疫苗能增强斑点鲖非特异性免疫功能,对鲖爱德华菌病起到一定的预防作用。     

Analysis of protective mechanisms in cultured ayu, Plecoglossus altivelis Temminck and Schlegel, administered Vibrio vaccine by the immersion method

Abstract. The protective effect of Vibrio angnillarum immunization administered by the direct immersion technique in ayu, Plecoglossus altivelis Temminck and Schlegel, was investigated. The activities of phagoeytic cells were suppressed at the time of vaccination by previous bath exposures to a cyclophosphamide solution containing a concentration of 500 mg/kg/ml on days 2 and 3 before vaccination. An artificial challenge was carried out 3 weeks after vaccination by bath exposure. The survival rate of the vaccine group pretreated with cyclophosphamide was 96%. In the group given traditional vaccine only, 73% survived; and in the non-vaccine control group, 10% survived. These results suggest that protective immunity of ayu vaccinated by the direct immersion technique may not implicate the early activities of phagoeytic cells.     

Immune responses of channel catfish, Ictalurus punctatus (Rafinesque), after oral or intraperitoneal vaccination with particulate or soluble Edwardsiella ictaluri antigen

Abstract. Groups of channel catfish, Ictalurus punctatus (Rafinesque). were administered Edwardsiella ictaluri twice in the form of whole outer membrane proteins (OMP) or heat-inactivated whole bacteria (IWB) orally or IWB intraperitoneally. Antibody titres and lysozyme concentrations were determined in serum, mucus, gut contents and gut washings taken 7, 14, 21 and 28 days after the second antigen administration. Bacterial killing by anterior kidney neutrophils was determined 7, 14 and 21 days after the second antigen administration. Enhanced killing of tumour targets by nonspecific cytotoxic cells (NCC) was determined 7 days after the second antigen administration. Serum antibody titres of all treatment groups were significantly increased above those of the control group throughout all sampling periods. Fish receiving oral administration of OMP or IWB exhibited maximum serum antibody titres on day 21 or 28. Antibody titres were also detected in mucus, gut washings and gut contents, but did not reach the level of those in the serum. Bacterial killing was significantly increased only on day 7, and could not be correlated to antibody titre or lysozyme concentration. Bacterial killing was found to be the result of a heat-labile serum factor. NCC activity of fish vaccinated with IWB orally was significantly higher than that of fish vaccinated with OMP orally or controls. Although intraperitoneal vaccination consistently produces higher titre antisera, the results of this study support the idea that oral vaccination can induce antibody systhesis.     

Protection of turbot, Scophthalmus maximus (L.), and rainbow trout, Oncorhynchus mykiss (Richardson), against vibriosis using two different vaccines

Abstact. The potency of a whole-cell bacterin (WCB) and a toxoid enriched whole-cell vaccine (WCEB) administered intraperitoncally into rainbow trout, Oncorhynchus mykiss (Richardson), were compared. The most effective vaccine was further evaluated by bathing turbot, Scophthalmus maximus (L.). These vaccines were composed of three strains of V. anguillarun , of the serotypes 01 and 02. Both vaccines conferred the highest protection against strains of serotype 01 within 4 weeks. With the toxoid enriched vaccine giving the best results (77 RPS). When trout were revaccinated after 7 weeks with this vaccine, good protection was achieved against strains of serotypes 01 and 02. Interestingly, when the WCEB was administered by bath to turbot, acceptable levels of protection against strains of both serotypes were obtained after 4 weeks of immunization.     

Survival and humoral antibody response of Atlantic salmon, Salmo salar L., vaccinated against Aeromonas salmonicida ssp. achromogenes

Atlantic salmon were vaccinated against Aeromonas salmonicida ssp. achromogenes (Asa) by injection with three vaccines developed in our laboratory and an autogenous bacterin (IcelandBiojec.OO, IBOO) produced by a commercial vaccine producer. The humoral antibody responses to bacterial antigens were monitored by ELISA and Western blotting. The fish were challenged by infection with Asa 6 and 12 weeks post-vaccination. Protection was induced in all groups of vaccinated fish. The protection achieved was time-dependent. The autogenous bacterin, IBOO, induced a protective immune response later than our experimental vaccines. All the vaccines tested induced specific antibody response that increased between 6 and 12 weeks after vaccination. The antibody response was mainly directed against the A-layer protein, but antibodies to other bacterial components were also detected. Significant correlation was obtained between the antibody titre to extracellular Asa antigens, induced by the different vaccine preparations, and survival of vaccinated fish challenged by a virulent Asa strain. Furthermore, the detection of antibodies directed against an extracellular toxic metallo-caseinase, AsaP1, in fish sera correlated with protection.     

Experimental Cryptobia salmositica (Kinetoplastida) infections in Atlantic salmon, Salmo salar L.: cell-mediated and humoral immune responses against the pathogenic and vaccine strains of the parasite

Hatchery-reared Atlantic salmon, Salmo salar L., were vaccinated intraperitoneally (i.p.) with a live attenuated Cryptobia salmositica vaccine (either 100 000 or 5000 parasites fish⁻¹) and 4 weeks later were challenged with the parasite (either 100 000 or 5000 parasites fish⁻¹). Unvaccinated, infected salmon had high parasitaemias and were anaemic. Fish given a high dose (100 000 parasites fish⁻¹) had higher parasitaemias than fish given the lower dose. Vaccinated fish had low parasitaemias and a mild anaemia, but recovered quickly after challenge. Complement-fixing antibody increased in vaccinated fish after challenge and was highest at 2 weeks post-challenge. The cell-mediated response (both T cells and B cells) was depressed in infected fish until 4 weeks after infection. In vaccinated fish, the humoral response (i.e. B-lymphocytes) was greater than the cell-mediated response (i.e. T-lymphocytes). In contrast, infected fish had a greater cell-mediated than humoral immune response.