Oral Vaccination of Channel Catfish against Enteric Septicemia of Catfish Using a Live Attenuated Edwardsiella ictaluri Isolate

Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most problematic bacterial disease affecting catfish aquaculture in the southeastern United States. Efforts to develop an effective ESC vaccine have had limited industrial success. In commercial settings, ESC vaccines are typically administered by immersion when fry are transferred from the hatchery to rearing ponds. While this approach is a practical method of mass delivery, this strategy administers vaccines to very young fish, which lack a fully developed immune system. To circumvent this limitation, an oral vaccination strategy was evaluated as a means of immunizing catfish at the fingerling stage of production, when fish possess a more complete immune arsenal. A virulent E. ictaluri isolate (S97-773) was attenuated by successive passage on media containing increasing concentrations of rifamycin. In laboratory trials, cultured vaccine was diluted and mixed with feed (100 mL diluted vaccine/454 g feed). This mixture was then fed to Channel Catfish Ictalurus punctatus fingerlings. Two separate dilutions of cultured vaccine (1:10 and 1:100) were used to create the vaccine–feed mixture, equating to estimated doses of 5 × 10⁷ and 5 × 10⁶ CFU/g of feed, respectively. After 30 d, catfish were exposed by immersion (1 × 10⁶ CFU/mL) to the virulent parental strain of E. ictaluri. The target dose (1:100 dilution, ～5 × 10⁶ CFU/g of feed) offered exceptional protection (relative percent survival = 82.6–100%). In addition, negligible deaths occurred in fish vaccinated at 10 times the target dose (1:10 dilution, ～5 × 10⁷ CFU/g of feed). In pond trials, antibody production increased 18-fold in orally vaccinated fish. When compared with nonvaccinated controls, vaccination significantly improved survival, feed fed, feed conversion, biomass produced, and total harvest. This research demonstrates Channel Catfish can be successfully immunized in a commercial setting against E. ictaluri with a single dose of an orally delivered, live attenuated, E. ictaluri vaccine.

Received July 31, 2014; accepted March 2, 2015     

DNA Vaccination Partially Protects Muskellunge against Viral Hemorrhagic Septicemia Virus (VHSV-IVb)

Abstract

A DNA vaccine containing the glycoprotein (G) gene of the North American viral hemorrhagic septicemia virus (VHSV) genotype IVb was developed to evaluate the immune response of fish following vaccination and evaluate its efficacy in protecting a susceptible species, the Muskellunge Esox masquinongy, against VHSV-IVb challenge. Seven weeks (539 degree-days) following vaccination with 10 μg of either pVHSivb-G or a control plasmid, Muskellunge were challenged by immersion with 10⁵ plaque-forming units (pfu)/mL of VHSV-IVb. Fish vaccinated with pVHSivb-G had a relative percent survival (RPS) of 45%. Vaccinated fish also had significantly lower mean viral titers in tissues (4.2 × 10² pfu/g) and viral prevalence (4%) than fish receiving the plasmid control vaccine (3.3 × 10⁵ pfu/g; 82%). Neutralizing antibodies were detected 28 d (308 degree-days) postchallenge (11 weeks postvaccination) in 100% of Muskellunge vaccinated with pVHSivb-G compared with only 12% of plasmid-control-vaccinated Muskellunge, suggesting robust induction of a secondary, adaptive immune response. In addition, pVHSivb-G–vaccinated Rainbow Trout Oncorhynchus mykiss challenged 7 d (100 degree-days) postvaccination with the heterologous novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), experienced an RPS of 61%, compared to control fish, suggesting induction of an early and transient nonspecific antiviral immune response. This study provides an important starting point for VHSV-IVb vaccine development and useful information about the antiviral immune response elicited by DNA vaccination in a nondomesticated fish species.

Received May 1, 2016; accepted September 1, 2016     

Communications: Protective Immunity Induced by a Commercial Vibrio Vaccine in Hybrid Striped Bass

Abstract

Commercial Vibrio anguiliarum-V. ordalii bacterin was used to vaccinate hybrid striped bass (Morone saxatilis ♀ × M. chrysops ♂) to test the vaccine efficacy against vibriosis. Vaccination by direct immersion of fish in diluted Vibrio vaccine for 20 s resulted in increased protective immunity. The relative percent survival of hybrid striped bass challenged 35 d after vaccination was 66.7% for those challenged by 1-h immersion exposure to 7.03 × 10⁷ V. anguillarum cells/mL, 75.0% for those challenged by injection with 3.51 × 10⁵ cells/fish, and 86.7% for those challenged by injection with 3.51 × 10⁴ cells/fish.     

Leptospira interrogans serovar pomona vaccines with different adjuvants in cattle

Three groups of four heifers were vaccinated twice, 11 weeks apart. One group received a commercial pomona vaccine with aluminium hydroxide adjuvant, the second a similar experimental vaccine, and the third a Freund's compete adjuvant (FCA) vaccine. After 47 weeks, the heifers were challenged with at least 65 × 10⁸ virulent serovar pomona organisms.

All vaccinated animals resisted the challenge, and leptospirae were only found in urine from unvaccinated controls.

The outcome of the challenge was not predictable from microscopic agglutination, cold and warm complement fixation, and growth inhibition titres.

The FCA vaccine gave rise to considerably higher antibody responses than the two aluminium hydroxide vaccines, which gave similar responses.     

Vaccination of Rainbow Trout against Infectious Hematopoietic Necrosis (IHN) by Using Attenuated Mutants Selected by Neutralizing Monoclonal Antibodies

Abstract

A neutralizing monoclonal antibody against infectious hematopoietic necrosis virus (IHNV) was used to select neutralization-resistant mutants from isolates of virus obtained from adult steelhead Oncorhynchus mykiss returning to the Round Butte Hatchery (RB mutants) on the Deschutes River in Oregon, USA, and from rainbow trout (nonanadromous O. mykiss) at a commercial hatchery in the Hagerman Valley of Idaho, USA (193-110 mutants). Two of the mutants, RB-1 and 193-110-4, were significantly (P < 0.001) attenuated compared with parental strains. Vaccination of rainbow trout by waterborne exposure to the mutants conferred solid protection against challenge with wild-type virus. In some trials, fish vaccinated with the RB-1 mutant at 50% tissue culture infectious doses (TCID50) of 1 × 10⁴–1 × 10⁵ TCID50/mL or with the 193-110-4 mutant at 1 × 10²–1 × 10³ TCID50/mL, held for 14 d, then challenged with the homologous wild-type strain at 1 × 10⁵ TCID50/mL showed relative percent survival of 95–100% (P < 0.005). There was no significant difference (P > 0.05) in protection among fish exposed to the RB-1 vaccine strain at a dose of 1 × 10⁵ TCID50/mL for periods of either 1, 12, or 24 h, held for 14 d, and then challenged with the wild-type RB isolate, although the 1-h exposure seemed to be somewhat less effective. Fish were vaccinated with the RB-1 strain at 1 × 10³–1 × 10⁵ TCID50/mL for 24 h then challenged after 1, 7, 14, or 21 d with the wild-type RB isolate. No significant (P > 0.1) protection was observed at 1 d postvaccination, but the relative percent survival increased progressively at each subsequent challenge period, becoming statistically significant by day 7 (P < 0.001) and beyond. These results suggested that resistance to challenge with wild-type virus resulted from development of IHNV-specific immunity and not from viral interference or interferon induction, and they reinforce the potential of an attenuated vaccine to control this important disease.     

Vaccination of cattle against brucellosis using either a reduced dose of strain 19 or one or two doses of 45/20 vaccine

SUMMARY Three groups, each of 14 mature Jersey heifers, were vaccinated. They were mated about 2 months later and those that became pregnant were challenged at about 6.5 months of pregnancy by the conjunctival application of virulent Brucella abortus. Group 1 heifers received 2 doses of B. abortus 45/20 vaccine 2 months apart. Only 5 of the 14 heifers became pregnant, and of these 5 only one resisted challenge. Group 2 heifers received only one dose of 45/20 vaccine, 5 of the 10 challenged resisted infection. Group 3 heifers received 3 × 10⁸ cfu of strain 19. Six of the 10 heifers challenged resisted infection. All of 5 non-vaccinated control cattle became infected. It appeared advantageous to give only one dose of 45/20 rather than 2 as presently recommended. A single dose of 45/20 vaccine induced resistance to virulent B. abortus approximately equal to that given by the reduced dose of strain 19. One dose of 45/20 vaccine stimulated transient serological positivity in 2 of 28 heifers whereas the reduced dose of strain 19 gave rise to persistent titres in 2 of 14 vaccinated heifers.     

Occurrence and Distribution of Aeromonas salmonicida Infections on Swedish Fish Farms, 1951–1987

Abstract

The occurrence of typical and atypical Aeromonas salmonicida infections on Swedish fish farms was surveyed, and the methods for controlling the disease are described here. This review is based on data for a period of 36 years from fish health control studies conducted by the National Veterinary Institute and the Salmon Research Institute. The epizootiology and routes of transmission of the pathogens are described, and policy and methods for disease control are discussed. The distribution of atypical A. salmonicida infections was entirely different from that of typical infections. There was also circumstantial evidence of different routes of transmission for typical and atypical A. salmonicida infections. Although subclinical carriers of atypical A. salmonicida occurred among Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss on fish farms, acute disease outbreaks were most frequent among Arctic char Salvelinus alpinus and brown trout Salmo trutta. On the other hand, acute outbreaks of typical A. salmonicida infections were frequent among Atlantic salmon and rainbow trout.     

Cell-Surface-Associated Properties of Fish Pathogenic Bacteria

Abstract

Pathogenicity assays showed that 33 of 42 potentially pathogenic strains of bacteria tested were virulent to rainbow trout Oncorhynchus mykiss. Regardless of their degree of virulence to fish, strains of motile Aeromonas, A. salmonicida, and Vibrio anguillarum were moderately hydrophobic. Only 46 and 25°10 of the strains were able to hemagglutinate human and trout erythrocytes, respectively. Hydrophobicity and hemagglutination were practically absent in isolates of Yersinia ruckeri. A notable number of the strains positively adhered to salmonid (51%) and nonsalmonid (55%) fish cells. Whereas the treatment of the bacteria with proteinase K or trypsin did not decrease the hydrophobicity of the isolates, within motile Aeromonas and A. salmonicida species, strains with both protease-sensitive and -resistant hemagglutinating and adhesive abilities occurred. The effects of heat and sugars on hemagglutinating and hydrophobic properties varied within all bacterial groups. Although treatment of strains with D-mannose or L-fucose had distinct effects on adhesiveness according to the bacterial species and the cell system used, none of the heat-treated (80°C for 15 min) bacteria lost their capacity to adhere to cultured fish cells. The results showed that there was no direct relationship between any of the cell surface properties analyzed and the degree of virulence of the strains.     

Effect of Endobronchial Challenge with Actinobacillus pleuropneumoniae Serotype 9 of Pigs Vaccinated with a Vaccine Containing Apx Toxins and Transferrin‐binding Proteins

The efficacy of a subunit vaccine containing the Apx toxins of Actinobacillus pleuropneumoniae and transferrin‐binding proteins was determined. Ten pigs were vaccinated twice with the vaccine. Eight control animals were injected twice with a saline solution. Three weeks after the second vaccination, all pigs were endobronchially inoculated with 10^6.5 colony‐forming units (CFU) of an A. pleuropneumoniae serotype 9 strain. In the vaccine group, none of the pigs died after inoculation. Only one pig of the control group survived challenge. Surviving pigs were killed at 7 days after challenge. The mean percentage of affected lung tissue was 64% in the control group and 17% in the vaccine group. Actinobacillus pleuroípneumoniae was isolated from the lungs of all animals. The mean bacterial titres of the caudal lung lobes were 5.0 × 10⁸ CFU/g in the control group and 3.0 × 10⁶ CFU/g in the vaccine group. It was concluded that the vaccine induced partial protection against severe challenge.     

Effect of booster shot and investigation of vaccination efficacy period against herpesviral haematopoietic necrosis (HVHN) in goldfish Carassius auratus

In this study, the efficacy period of an intraperitoneal vaccination and effect of a booster shot of vaccine against herpesviral haematopoietic necrosis (HVHN) in goldfish Carassius auratus were investigated. Cell culture supernatant of cyprinid herpesvirus 2 (CyHV-2), causative agent of HVHN, propagated in goldfish fin (GFF) cells was inactivated with formalin (0.1%, v/v) for 2 days at 4 °C. Three groups of the variety Ryukin were individually intraperitoneally injected with the vaccine and each group was separately maintained in replicate tanks. After 4 weeks (Vaccinated-4w-1 and 2) and 8 weeks (Vaccinated-8w-1 and 2) from the first vaccination, the fish were CyHV-2-challenged by the immersion route (10 TCID₅₀ l⁻¹). In addition, the other vaccinated group of fish were injected with a booster vaccine 4 weeks after the first vaccination as the Vaccinated-booster groups, then the fish of these groups were CyHV-2-challenged by the immersion route (10 TCID₅₀ l⁻¹) after 8 weeks from the first vaccination. The mean of the relative percentage survival (RPS) values of the Vaccinated-4w and 8w groups showed 42.5% and 57.6%, respectively. In addition, the mean RPS value of Vaccinated-booster groups showed 63.6%. Statistical analysis showed significantly higher survival rates in all the vaccinated groups than those of the respective negative control groups using Fisher's exact test. Moreover, the survival rates of vaccinated-booster groups were significantly higher (p = 0.036) compared with the respective control groups by Student's t test. The present study shows the efficacy period of the vaccine is at least 8 weeks and a booster shot showed a tendency to enhance the protection against HVHN in goldfish.     

In Vitro Antimicrobial Activity of Sarafloxacin against Clinical Isolates of Bacteria Pathogenic to Fish

Abstract

An in vitro susceptibility assay of sarafloxacin (A-56620), a new 4-quinolone, was performed against five important bacterial species that are pathogenic to fish. A collection of 44 clinical isolates and five corresponding type strains were included in the study. The objectives were to determine the minimal inhibitory concentrations (MICs) of sarafloxacin by a drug microdilution method and to compare the MIC values at two different temperatures, 4 and 15°C. Sarafloxacin was active against all species tested and showed the following mean MIC values at 15 and 4°C, respectively, against the bacterial pathogens investigated: Aeromonas salmonicida subspecies salmonicida, 0.029 and 0.045 μg/mL; atypical A. salmonicida, 0.053 and 0.041 μg/mL; Vibrio anguillarum, 0.085 and 0.054 μg/mL; V. salmonicida, 0.125 and 0.123 μg/mL; and Yersinia ruckeri, 0.023 and 0.031 μg/mL. The MICs ranged from 0.0025 μg/mL (or less) for two strains of A. salmonicida salmonicida to 0.32 μg/mL for one strain of atypical A. salmonicida and one strain of V anguillarum. A decrease in antimicrobial activity was observed as the incubation temperature was lowered from 15 to 4°C; however, no significant statistical difference between the measured values was demonstrated.     

Efficacy of lipopolysaccharide antigen of Yersinia ruckeri in rainbow trout by intraperitoneal and bath immersion administration

In this study, Intraperitoneal (IP) and bath immersion (BI) vaccine trials were conducted in fish with a mean weight of 6.3 g. Rainbow trout vaccinated with lipopolysaccharide (LPS) was 50 mg/L protein concentration and challenged by IP injection with 9.8 × 10⁶ cell/ml of Yersinia ruckeri at 45 days post-immunization had a relative percent survival (RPS). To obtain an effective bath immersion vaccine against yersiniosis, LPS preparation was obtained from the Y. ruckeri and with the LPS antigen. After 28 and 60 days vaccinated fish with first and second immunizations by LPS were challenged via intraperitoneal injection with 9.8 × 10⁶ cell/ml of Y. ruckeri for evaluating the mortality rates and calculating the relative percentage of survival (RPS). RPS value of experimental groups, which was significantly (P < 0.05) larger than that of the control group.     

Physiological and Immunological Effects of Adjuvanted Aeromonas salmonicida Vaccines on Juvenile Rainbow Trout

Abstract

The effects of injectable vaccines against Aeromonas salmonicida on oxygen consumption, growth, kidney lysozyme activity, and anti-A. salmonicida plasma antibody titers of juvenile rainbow trout Oncorhynchus mykiss were examined. The vaccines were A. salmonicida bacterin only, bacterin adjuvanted with levamisole, bacterin in emulsified oil, microencapsulated bacterin, microencapsulated bacterin with muramyl dipeptide, microencapsulated bacterin with β-1,3-glucan, and microencapsulated bacterin with Vibrio anguillarum lipopolysaccharide (LPS). The greatest and broadest ranges of responses were caused by the microencapsulated bacterin with V. anguillarum LPS. Oxygen consumption rates and specific growth rates were significantly higher over the course of 1 month among fish treated with the LPS vaccine. These fish also maintained a higher anti-A. salmonicida plasma antibody titer and kidney lysozyme activity for a substantially longer period than fish receiving the other treatments.     

Comparative protection of two different commercial vaccines against Yersinia ruckeri serotype O1 and biotype 2 in rainbow trout (Oncorhynchus mykiss)

Differentially extended specific protection by two commercial vaccines against Yersinia ruckeri serotype O1 biotype 2 was studied following 30 s immersion exposure. Rainbow trout were challenged intra-peritoneally (i.p.) with Y. ruckeri serotype O1, biotype 2 (≈10⁶ to 10⁷ CFU/fish) at 4, 6 and 8 months after vaccination with vaccines containing either biotype 1 (AquaVac^® ERM) or both biotypes 1 and 2 (AquaVac^® RELERA?). The specific pattern of vaccine-mediated protection was evaluated by relative percentage survival (RPS) analysis at 4 and 6 months post-vaccination and by obtaining gross pathological observations at 4 and 8 months respectively. We determined specific significant and superior protection in terms of increased survivability in AquaVac^® RELERA? vaccinated fish and observed correspondingly fewer pathological changes. The challenge trials indicated a longer protection for at least 6 months without any booster vaccination. A specific and adaptive response induced by AquaVac^® RELERA? vaccine against Y. ruckeri biotype 2 was clearly indicated. In addition, some degree of cross protection rendered by AquaVac^® ERM containing biotype 1 during infection with Y. ruckeri biotype 2 was also noted.     

The Effect of High Total Ammonia Concentration on the Survival of Channel Catfish Experimentally Infected with Flavobacterium columnare

Abstract

Ammonia concentrations in water can affect the severity of Flavobacterium columnare infections in fish. Two trials lasting 7 d each were conducted to determine the effect of a single immersion flush treatment of total ammonia nitrogen (TAN; 15 mg/L) on the survival of channel catfish Ictalurus punctatus infected with F. columnare; the chemical was added while the water flowed continuously through the tanks. Both trials consisted of four treatments: (1) no ammonia exposure and no bacterial challenge (control), (2) ammonia exposure only, (3) bacterial challenge only, and (4) both ammonia exposure and bacterial challenge. Two hours after exposure to ammonia, the highest un-ionized ammonia level was 0.43 mg/L. The percent un-ionized ammonia is based on TAN, temperature, and pH. Caudal fins from three fish in each treatment were sampled at 24 h posttreatment to be analyzed by quantitative real-time polymerase chain reaction (qPCR). No significant difference in survival (mean ± SE) was noted between the channel catfish in treatment 1 (95.2 ± 1.2%) and those in treatment 2 (95.6 ± 1.0%); however, survival in both treatments 1 and 2 differed significantly from that in treatments 3 (8.5 ± 4.5%) and 4 (41.8 ± 12.7%). Treatment 4 catfish had significantly higher survival than treatment 3 catfish. Quantitative PCR data showed that treatment 4 fish had significantly less F. columnare (7.6 × 10⁵) than did treatment 3 fish (1.2 × 10⁷), and treatment 2 fish (8.5 × 10³) had significantly less bacteria than did treatment 1 fish (6.9 × 10⁴), indicating that ammonia limited the F. columnare infection. The highest mean concentration of the bacteria (3.9 × 10⁷) was found on moribund fish. The ammonia concentrations tested did not negatively influence fish survival but interfered with the infection process. An in vitro assay was also conducted to evaluate the direct effects of ammonia on F. columnare.

Received September 15, 2010; accepted May 7, 2011     

Consequence of Cryptosporidiosis on the immune response of vaccinated broiler chickens against Newcastle disease and/or avian influenza

The consequence of cryptosporidiosis on the immune response of vaccinated chickens against Newcastle disease and/or avian influenza was studied by using 240, 1 day old, male, white Hy-Line chicks and divided into 8 groups and subgroups. Each group or subgroup was consisting of 30 chicks (15?×?2 replicates). The first and second groups were kept as unvaccinated control, G1uninfected and G2 infected. G3, G4 and G5 contained 2 subgroups A&B (G3A, G3B, G4A, G4B, G5A and G5B). Chicks of subgroup A were vaccinated only while chicks of subgroup B were infected and vaccinated. These chicks were orally inoculated with 5?×?10⁵ oocysts of Cryptosporidium baileyi (C. baileyi) at 2 days of age. Chickens were vaccinated intraocular with live Newcastle disease (ND) vaccine (Hitchner on day 7th and LaSota on day 17th of chicken life) (G3) or vaccinated by subcutaneous route with Volvac®- H5N2- AI vaccine on day 10 of chicken life (G4). Last group (G5) was infected similarly and vaccinated with ND and AI vaccines with the same day, dose and route of vaccination for each one. Random blood samples were collected for 3 weeks post-vaccination for investigation of humoral immune response against Newcastle and/or avian influenza vaccines by the haemagglutination inhibition (HI) test. The results showed that H5N2 vaccine at day 10 of chicken life is effective in chickens indicated by the geometric mean of HI titer against AI virus. The findings of this study showed that the infection with Cryptosporidia in the broiler chicken has a depressive effect on the immune status of the birds vaccinated against ND and/or AI vaccination. Moreover, the obtained protection rates against challenge with virulent ND virus observed to be parallel to the results of HI- test. Also, by using 2 different antigens (one commercial and field prepared antigen) to avian influenza virus, lower Geometric mean (GM) HI titer were appeared in infected and vaccinated group than vaccinated group only. A study of the relative lymphoid organs weight such as bursa of Fabricius from the experimental chicks indicated that those organs were comparable between the groups infected-vaccinated and vaccinated only. Non significant variations in final live weight between uninfected control and infected groups were indicated. Also, H5N2-AI vaccination at 10 days old did not affect the final live weight. ND and/or AI Vaccination could not be a substitute to application of good hygienic measures and fecal examination of the birds especially for protozoal diseases such as cryptosporidiosis. It could be concluded that cryptosporidiosis could be one cause of ND and/or AI vaccination failure in poultry farms.     

Systemic mycoses of two cats

Calves vaccinated twice with one commercial pomona vaccine had serum MAT titres up to 1:300, warm CFT titres of 1:20 and cold CFT titres of up to 1:80. A second commercial vaccine did not elicit any detectable antibodies.

All calves, vaccinated as well as unvaccinated, showed a rapid rise in antibody levels, detectable with all three tests, following challenge with 38 × 108 pomona organisms.     

Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White‐tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock and the cause for many faltering bovine tuberculosis eradication programmes. One approach in dealing with wildlife reservoirs of disease is to interrupt inter‐species and intraspecies transmission through vaccination of deer or cattle. To evaluate the efficacy of BCG vaccination in white‐tailed deer, 35 deer were assigned to one of three groups; one s.c. dose of 10⁷ CFU of M. bovis BCG Pasteur (n = 12); 1 s.c. dose of 10⁷ CFU of M. bovis BCG Danish (n = 11); or unvaccinated deer (n = 12). After vaccination, deer were inoculated intratonsilarly with virulent M. bovis. Lesion severity scores of the medial retropharyngeal lymph node, as well as all lymph nodes combined, were reduced in vaccinated deer compared to unvaccinated deer. BCG Danish vaccinated deer had no late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid‐fast bacilli compared to BCG Pasteur vaccinated or unvaccinated deer where such lesions were present. Both BCG strains were isolated as late as 250 days after vaccination from deer that were vaccinated but not challenged. In white‐tailed deer, BCG provides protection against challenge with virulent M. bovis. Issues related to vaccine persistence, safety and shedding remain to be further investigated.     

Safety of Strontium Chloride as a Skeletal Marking Agent for Pacific Salmon

Abstract

The purpose of this study was to evaluate the biological effects associated with administering strontium chloride as a marking agent to age-0 Chinook Salmon Oncorhynchus tshawytscha fry. Fish were held in a 0× (0 mg/L), 1× (3,000 mg/L; current standard dosage), 3× (9,000 mg/L), or 5× (15,000 mg/L) solution of strontium chloride for 72 h (three times the standard duration of 24 h). The mortality among fish in the 5× strontium chloride exposure group was significantly higher than that observed in the other groups. A dose-related effect on general fish behavior and on feeding behavior was observed. Fish in all test tanks appeared to feed to satiation, except for fish in the 5× tanks during days 2 and 3. Fish in all other test tanks behaved normally. No dose-related effect on fish growth was detected. Histopathological evaluations showed that fish in the 5× exposure group had a significantly higher number of gill lesions than the 0× group. Our mortality, behavioral, and histological assessments suggested that juvenile Chinook Salmon could be safely immersed for three consecutive days in a 9,000-mg/L solution of strontium chloride. This finding potentially expands the present 1,000–3,000-mg/L dosage and 24-h holding period that can be used to mark juvenile fish with strontium chloride solutions. The research also provides necessary target animal safety data for U.S. Food and Drug Administration approval of strontium chloride as an alternative marking method that is suitable for fish with a short holding time.

Received February 19, 2017; accepted July 16, 2017 Published online September 22, 2017