The reaction of the immune system of fish to vaccination: development of immunological memory in carp, Cyprinus carpio L., following direct immersion in Aeromonas hydrophila bacterin

Abstract. The development of immunological memory was investigated in carp following direct immersion in Aeromonas hydrophila bacterin. A single bath did not induce an increase in serum antibody (Ab) levels. A second bath at 1, 3 or 8 months resulted in clear secondary Ab responses. The highest Ab levels were reached in the group boosted after 3 months. However, at 12 months a second bath gave no further significant response. When a booster was given at the same intervals by intramuscular injection, instead of immersion, the induced memory was not expressed as a clear secondary response. It is concluded that direct immersion in a bacterial antigen did induce immunological memory. The duration of this slowly developing memory is limited in time (less than 12 months) and was only demonstrated following a second immersion. This last feature suggests the existence of a local or mucosal activity during the immune response after bath vaccination.     

Modification of the hyperosmotic infiltration method of vaccination against vibriosis in cultured ayu, Plecoglossus altivelis Temminck and Schlegel

Abstract. A vaccine solution of a formalin-killed culture of Vibrio anguillarum cells was observed to be toxic to young ayu when administered by the hyperosmotic infiltration method. The toxin was present in the culture broth. After the toxin was removed from the broth by centrifugation, the fish were dipped in 5.32% NaCl solution for 2 min and then in a solution containing precipitated cells for 3 min. The immunized fish were protected against vibriosis when challenged one month after immersion. The bacterin was administered to ayu by a further two methods, both using lyophilized whole cells of formalin-killed V. anguillarum. In one method, the fish were placed in a 5.32% NaCl solution for 2 min and then in a solution containing lyophilized cells at 2 g/l of well water for 3 min (two-step immersion). In the other method, the fish were placed in a 5.32% NaCl solution containing lyophilized cells also at 2 g/l for 3 min (one-step immersion). A high level of protection against artificial challenge was achieved with either method. No agglutinating antibodies to V. anguillarum were detected in either the serum or mucus of fish dipped in a vaccine solution, a supernatant, or a precipitated solution, one month after immersion. On the other hand, serum titres were detected in fish vaccinated by injection, although no titres were detected in mucus. LD₅₀ values are presented for the virulence of the V. anguillarum strain. Compared to the original strain, virulence increased after the third passage in ayu, but decreased after the thirteenth passage in medium.     

Onset of immunity in salmonid fry vaccinated by direct immersion in Vibrio anguillarum and Yersinia ruckeri bacterins

Abstract. The fry of several salmonid species were vaccinated by direct immersion in either Yersinia ruckeri or Vibrio anguillarum bacterin and the level of protective immunity was determined by the survival of fish after bath challenge with virulent organisms. The immersion time for effective vaccination was obtained within 5 s and protective immunity was demonstrated within 5 days at 18°C and within 10 days at 10°C. The minimum size at which maximum protective immunity occurred was between 1.0 and 2.5 g. Immunity appeared to be a function of size and not age, but differences in response among several species were indicated. In fish under 1.0 g, the level of protective immunity could be increased using a more concentrated bacterin. The results were similar with both bacterins.     

Duration of protective immunity and antibody titres measured by ELISA after vaccination of rainbow trout, Salmo gairdneri Richardson, against vibriosis

Abstract. The level of protective immunity in rainbow trout, Salmo gairdneri Richardson, following vaccination by intraperitoneal (i.p.) injection or by direct immersion with Vibrio anguillarum was studied over a period of 46 weeks. Antibody titres to V. anguillarum O-antigen were determined with ELISA at the time of each experimental infection to study the relation between specific antibody production and protective immunity. Both vaccination by i.p. injection and vaccination by direct immersion induced a high degree of protective immunity against vibriosis which lasted for at least 46 weeks. At each experimental infection, the proportions of seropositive fish were lower than the proportions of fish with protective immunity. However, seropositive individuals were found in both experimental groups throughout the study.     

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。     

A histological study of the response to challenge with vibriosis in ayu, Plecoglossus altivelis Temminck and Schlegel, vaccinated by immersion and injection with Vibrio anguillarum bacterin

Abstract. The histological responses of ayu, Plecoglossus altivelis , given an intramuscular injection of a formalin-killed bacterin of Vibrio anguillarum are described. Lesions at the site of injection showed muscular necrosis and infiltration of inflammatory cells by the second day after injection, and production of granulation tissue from the fifth to the fourteenth day. Protective responses against vibriosis were studied histopathologically in ayu that were vaccinated by intramuscular injection with formalin-killed Vibrio bacterin and by immersion in sonicated Vibrio bacterin, and challenged by a subcutaneous injection with viable Vibrio on the fourteenth day after the vaccination.
Efficacy of both methods was confirmed by the survival of vaccinated fish after the challenge. There was slight bacterial multiplication in the fish, and bacterial phagocytosis by infiltrated neutrophils and tissue necrosis in the injected area on the third day after the challenge. In contrast, non-vaccinated fish died within 24h of challenge, with extensive bacterial multiplication and tissue necrosis in the injected area and visceral organs.     

Oral vaccination trials with crayfish, Procambarus clarkii, to induce resistance to the white spot syndrome virus

The potential of oral vaccination against white spot syndrome virus (WSSV) in crayfish Procambarus clarkii was investigated. The protective effect of binary ethylenimine (BEI)-inactivated WSSV was tested by oral vaccination, followed by an oral challenge with WSSV. The crayfish fed with feed pellets coated with BEI-inactivated WSSV showed a resistance to WSSV on the seventh day post vaccination (dpv). The relative percentage survival values were 60%, 70% and 75% for the vaccinated once, twice and thrice with inactivated WSSV. Following an intramuscular injection experiment, no mortality was recorded in the inactivated WSSV group and the negative control at 17 days post challenge. The cumulative mortalities in the heated WSSV group and WSSV group were 100%. Shrimp that survived the WSSV challenge on the seventh day after cessation of oral vaccination were positive for the presence of WSSV by a polymerase chain reaction assay specific for WSSV. This result indicated that inactivated WSSV could protect crayfish against WSSV by oral delivery.     

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.     

Secondary immune response of rainbow trout following repeated immersion vaccination

Teleosts are able to raise a protective immune response, comprising both innate and adaptive elements, against various pathogens. This is the basis for a widespread use of vaccines, administered as injection or immersion, in the aquaculture industry. It has been described that repeated injection vaccination of fish raises a secondary immune response, consisting of rapid, accelerated and increased antibody reaction. This study reports how rainbow trout responds to repeated immersion vaccination against yersiniosis (ERM) caused by the bacterial pathogen Yersinia ruckeri. It was found that rainbow trout does not raise a classical secondary response following repeated immersion vaccination. Serum antibody titres were merely slightly increased even after three immunizations, using 30‐s immersion into a bacterin consisting of formalin‐inactivated Y. ruckeri (serotype O1, biotypes 1 and 2), performed over a 3‐month period. The densities of IgM‐positive lymphocytes in spleen of fish immunized three times were increased compared to control fish, but no general trend for an increase with the number of immunizations was noted. The lack of a classical secondary response following repeated immersion vaccination may partly be explained by limited uptake of antigen by immersion compared to injection.     

Hormone injections enhance the tolerance of land-locked ayu spermatozoa to cryopreservation

We evaluated the effects of maturation-stimulating hormones on the post-thaw motility of land-locked form ayu ( Plecoglossus altivelis ) spermatozoa. Male ayu were administered three intraperitoneal injections of either salmon pituitary extract (SPE; 0.2 or 0.6 mg g⁻¹ BW day⁻¹) or of 17, 20β-dihydroxy-4-pregnen-3-one (DHP; 2 or 10 μg g⁻¹ BW day⁻¹), the maturation-inducing steroid (MIS) in ayu. Before cryopreservation, the motility of spermatozoa of the SPE- and DHP-treated groups was significantly higher than that of the control group. Similarly, the comparative post-thaw motility (presented as a percentage of the motility obtained before cryopreservation) was significantly higher in the SPE group than in the control; however, there was no significant difference between the DHP group and the control. The effect of SPE and DHP on pre- and post-cryopreservation motility was not dose dependent. Our results suggest that the hormone(s) present in salmon pituitary are effective in enhancing the tolerance of ayu sperm cells to cryopreservation and that the MIS (DHP) is not involved in this process.     

The immune response of Atlantic salmon, Salmo salar L., to the causative agent of bacterial kidney disease, Renibacterium salmoninarum

Abstract. Both under-yearling and post-yearling Atlantic salmon parr produced high agglutinating antibody titres in response to a single intraperitoneal injection of killed bacterial kidney disease (BKD) cells emulsified in. Freund's complete adjuvant (FCA), Low or no response was observed in animals injected with BKD cells in saline or in animals vaccinated by hyperosmotic immersion. Immunological duration was insufficient in fish vaccinated as under-yearling parr to provide protective immunity 2 years later when the fish had become smolts. Atlantic salmon post-yearling parr injected with BKD cells in FCA demonstrated a reduced prevalence of BKD lesions compared to control animals when both were observed as smolts 1 year after vaccination.     

Inhibition of the attachment of Flavobacterium branchiophilum to the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

Preliminary experiments are described which aimed to identify compounds that could inhibit the attachment of Flavobacterium branchiophilum strains LAB4a and ATCC 35035 to the gills of rainbow trout. Total inhibition was never achieved, regardless of the compound tested. Formalin-killed or acetone-killed F. branchiophilum cells retained at least some of their adherent nature, relative to untreated (live) cells. Adherence was reduced by 22–33% following immersion of fish in one litre of water containing 0.21 mg of a homologous crude fimbrial extract. When fish were immersed in water containing hyperimmune rainbow trout antiLAB4a serum, a dose-dependent decrease in attachment (a reduction of 15% to 63%) of LAB4a to the gills was observed. Rainbow trout anti-LAB4a serum also reduced the attachment of ATCC 35035 to the gills, but this reduction was not significant. Adherence of LAB4a was not inhibited following exposure of fish to group 1 carbohydrates (arabinose, mannose and xylose), group 2 carbohydrates (dextrose, galactose and lactose), group 3 carbohydrates (galactosamine, glucosamine and fucose) or group 4 carbohydrates (N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-neuraminic acid and the globoceramide glycolipid from human erythrocytes). In contrast, when rainbow trout erythrocytes were incubated with the bacteria prior to bath challenge, this resulted in an 87% and 53% reduction in gill-associated LAB4a and ATCC 35035 antigen, respectively, following immersion of rainbow trout in this suspension.     

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.     

Growth of Flavobacterium psychrophilum in fish serum correlates with pathogenicity

Flavobacterium psychrophilum isolates, obtained from ayu, Plecoglossus altivelis, three species of salmonids and two species of cyprinids in Japan, were used in this study. Bacteria were inoculated to serum prepared from ayu or red spotted masu trout (RSMT), Oncorhynchus masou ishikawae, and incubated at 18 °C for 24 h. All isolates (n = 19) from ayu grew well with a 9- to 116-fold increase of CFU in ayu serum, while CFU decreased markedly in RSMT serum. In contrast, isolates (n = 17) from fish species other than ayu exhibited no growth in ayu serum, but some isolates from salmonids survived or grew (1.2-23.5 fold increase of CFU) in RSMT serum. The isolates that could not survive or grow in ayu and RSMT sera grew well in both heat-inactivated sera of ayu and RSMT. Experimental infection by intraperitoneal injection showed that ayu isolates examined were all pathogenic to ayu but not to RSMT, while none of the isolates from salmonids and cyprinids were pathogenic to ayu but some showed pathogenicity to RSMT. These results indicate that the in vitro growth ability of F. psychrophilum isolates in fish serum correlates well with their pathogenicity to fish, particularly in ayu.     

Effects of Feeding Rate and Feeding Frequency on Survival, Growth, and Body Composition of Ayu Post-Larvae Plecoglossus altivelis

The effects of feeding rate and feeding frequency on survival, growth and body composition of ayu post‐larvae (0.15 g in body weight and 3.5 cm in total length) were investigated in this study. A factorial experimental design of two feeding rates (3 and 6% of body weight of fish per meal) five feeding frequencies (one meal in 2 d, one meal a day, two meals a day, four meals a day, and six meals a day) with three replicates was used. Survival of ayu post‐larvae was significantly (P 0.05) affected by feeding frequency but not by feeding rate. Survival of ayu improved linearly with feeding frequency at both feeding rates. Weight and length gains and specific growth rate (SGR) of ayu was significantly (P 0.05) affected by feeding frequency but not by feeding rate, with weight and length gains and SGR linearly elevated with increasing feeding frequency at both feeding rates. The greatest weight and length gains were observed in fish receiving six meals daily at both feeding rates; however, no significant difference in weight gain was observed among two, four, and six meals a day, or in length gain between four and six meals a day. Feed efficiency ratio (FER) was significantly (P 0.05) affected by both feeding rate and feeding frequency. FER linearly decreased with feeding frequency at both feeding rates or feeding rate in the same feeding frequency. When the total daily amount of feed supply was constant with various feeding frequencies at different feeding rates (one meal a day at 3% feeding rate and one meal in 2 d at 6% feeding rate, two meals a day at 3% feeding rate and one meal a day at 6% feeding rate, or four meals a day at 3% feeding rate and two meals a day at 6% feeding rate), improvement in survival, weight and length gains, and SGR was observed in fish with higher feeding frequency at lower feeding rate. Moisture, protein, and lipid content of fish were not significantly (P > 0.05) affected by either feeding rate or feeding frequency. However, lipid content of ayu linearly increased with feeding frequency at 6% feeding rate. The highest body lipid content was observed in fish receiving six meals daily at both feeding rates. Ash content of fish was significantly (P 0.05) affected by feeding frequency but not by feeding rate. Based on performance of ayu, it can be concluded that optimum feeding rate and feeding frequency for ayu post‐larvae (an initial weight of 0.15 g) were 3% per meal and four meals a day, respectively, under these experimental conditions.     

Studies on Vaccination of Channel Catfish,Ictalurus punctatus,Against Edwardsiella ictaluri

Enteric septicemia of cattish (ESC), caused by the bacterium Edwardsiella ictaluri, has become the most significant disease problem affecting the commercial channel catfish, Ictalurus punctatus, industry in the United States. Although antibiotics are used extensively for the control of ESC, there are inherent problems associated with their use. Consequently, experiments were initiated to evaluate the effectiveness of vaccination program that used immersion and oral delivery methods to administer a killed E. ictaluri vaccine to fry and fingerling channel cafish. In a preliminary pond study with laboratory challenge, mortality in a group vaccinated with a combination of immersion and oral procedures was only 5.0% in both high- and low-dose challenges. This was significantly different (P c 0.01) from non-vaccinated controls, which had 46.7%mortality in the lowdose challenge and the 6 1.7% mortality in the highdose challenge. This corresponds to relative percent survival (RPS) values of 89.3 and 91.9 respectively. Subsequent field trials further indicated the efficacy of a vaccination program for the prevention of ESC in channel catfish. In 1987-1988, a field study was conducted using 12 commercial ponds, with three replicates of four treatments. The four treatments included vaccination by immersion only, oral only, a combination of both immersion and oral procedures, and non-vaccinated conwols. Relative percent survival was 57.4 for the immersion only treatment, 50.3 for the oral only treatment, and 53.5 for the combination immersion and oral treatment. In 1989-1990, no significant difference was found between vaccinated and non-vaccinated fish. However, in 1989-1990, a vaccine-oil emulsion was topcoated on a floating feed, rather than incorporating vaccine in a sinking pellet. In 1990-1991, overall mortality in vaccinated fish was significantly less (P < 0.05) than non-vaccinated fish, with 41.2% mortality in vaccinates compared to 63.5% in non-vaccinated fish, for an RPS of 35.1. In examining RPS values for individual farms, two farms had excellent results, with RPS values of 81.3 and 76.9; two farms had only moderate success, with RPS values of 26.6 and 15.4; and one location had greater mortality in the vaccinated fish than in the non-vaccinated fish. However, that farm had only two ponds in the study and experienced significant losses to proliferative gill disease in the pond with vaccinated fish.     

Ultrasonic immunization of sea bream, Pagrus major (Temminck & Schlegel), with a mixed vaccine against Vibrio alginolyticus and V. anguillarum

In order to clarify the effectiveness of ultrasonication on vaccine delivery, juvenile sea bream, Pagrus major , were treated with eight different ultrasonic methods. A mixed vaccine against Vibrio alginolyticus and V. anguillarum was used to immunize the fish . The intensity and frequency of the ultrasound were 280 mW cm^–2 and 35 kHz, respectively. The ultrasonic methods included continuous or pulsed ultrasound for 3 min, and continuous or pulsed ultrasound for 3 min before and/or after immersion for 3 min. Of all the eight ultrasonic methods tested, `pulsed ultrasound followed by immersion' and `immersion, pulsed ultrasound, and followed by immersion again' provided the best protection, which were comparable with protection of fish immunized by intraperitoneal injection. Moreover, the convenience of applying these two ultrasonic methods for immunization was comparable with the immersion method and was much better than intraperitoneal injection. If 2 × 10⁸ CFU mL^–1 of this mixed vaccine was used for vaccination repeatedly five times by ultrasonic methods, it could still produce good protection for the immunized sea bream. Therefore, the ultrasonic method is an effective and practical approach for fish vaccination on a large scale.     

Improved recapture rate of vaccinated sea-ranched Atlantic salmon, Salmo salar L.

Vaccination of sea-ranched Atlantic salmon was conducted in order to investigate if immuno-prophylactic measures could improve their survival. Fish were either vaccinated by bath or injection. A total of 66 000 fish were reared in fresh water at a hatchery on the island of Bornholm and at the presmolt stage were separated in three groups each comprising of 22 000 fish. One group was vaccinated intraperitoneally with a polyvalent vaccine (containing killed Vibrio anguillarum serotype O1 and O2, Yersinia ruckeri and Aeromonas salmonicida ). A second group was bath vaccinated with the corresponding vaccine-components and the third group was used as a non-vaccinated control. One month after vaccination these groups were allocated to three separate net-pens located 500 m from the coastline of the island. After 4 months in the net-pens, 1000 fish from each cage were tagged with Carlin-tags below the dorsal fin. The fish were then released for a migration period in the Baltic Sea. Following a sea period of 40 months (45 months post-vaccination), the recapture rates of the groups were calculated from the returned tags from fishermen. Recapture of the injection vaccinated group was significantly higher (25%) compared with the bath vaccinated fish (14.7%) and the control group (16.8%).     

Turbid water induces refuge behaviour of a commercially important ayu: A field experiment for interstream movement using multiple artificial streams

Riverine systems are inherently dendritic in nature, and turbid water may not necessarily flow equally throughout an entire catchment. Tributaries have the potential to serve as refugia from temporarily turbid water in the main stem. As ayu (Plecoglossus altivelis) are one of the important species for the inland fisheries industry and recreational fishing, their behaviour in a branched river network is a primary concern. We released ayu with radio‐transmitting tags into two experimental streams that joined at a downstream pool. Turbid water was released into one of the streams, and we identified whether avoidance movement of ayu between the two experimental streams was triggered by flowing turbid water. The suspended solid concentration elevated rapidly after adding the turbid water and was maintained at more than 400 mg/L for 3 hr. Avoidance movement of ayu was triggered when the suspended solid concentration exceeded approximately 200 mg/L. Then, most ayu moved from the turbid stream to the nonturbid one, leading to a difference in ayu density between the two streams. Therefore, we demonstrated that turbid water induced interstream movement of a commercially important ayu as refuge behaviour, suggesting that river connectivity is important for the persistence of the ayu.