Experimental Lactococcus garvieae infection in rainbow trout,Oncorhynchus mykiss,Walbaum 1792: a comparative histopathological and immunohistochemical study

The aim of this study was to induce Lactococcus garvieae infection in young and adult fish through different routes [intraperitoneal (IP) and immersion (IM)] and to investigate the pathogenesis and histopathological and immunohistochemical findings comparatively. For this purpose, a total of 180 rainbow trout (90 young, 20 ± 5 g and 90 adult, 80 ± 10 g) obtained from a commercial fish farm were used. The fish were divided into eight groups, four experimental groups (Young‐Adult IP groups and Young‐Adult IM groups, each contain 30 fish) and four control groups (Young‐Adult IP Control groups and Young‐Adult IM control groups, each contain 15 fishes). The experimental study was conducted using L. garvieae, and confirmatory identification was performed by PCR. The sequence result of the PCR amplicon of 16S rDNA from isolate L. garvieae LAC1 was determined and deposited in the GenBank database under accession number KC883976 . Fish in the IP groups were intraperitoneally administered an inoculate containing 10⁶ cfu mL^?1 bacteria 0.1 mL. In the IM groups, fish were kept in inoculated water containing 10⁸ cfu mL^?1 bacteria for 20 min. Mortality as well as clinical and pathological findings was recorded daily, and significant differences in macroscopic and microscopic results were observed between the IP and IM administration groups. All tissue samples were immunohistochemically stained by the avidin‐biotin‐peroxidase complex and immunofluorescence (IF) methods using polyclonal antibody to detect L. garvieae antigens. In immunoperoxidase staining in the IP groups, positive reactions to bacterial antigens were most commonly seen in the spleen, kidney, heart, liver, peritoneum and swim bladder. In the IM groups, bacterial antigens were most commonly found in the eye, gill, spleen and kidney. In the IF method, the distribution of antigens in tissue and organs was similar to the reactions with immunoperoxidase staining. Finally, in this experimental study, an important correlation was seen between the distribution of L. garvieae antigens and lesions developing in many organ and tissues.     

Induction and characterization of a lysogenic bacteriophage of Lactococcus garvieae isolated from marine fish species

This study investigated the presence of prophages in Lactococcus garvieae isolated from several marine fish species in Japan. Representative strains of 16 bacterial genotypes (S1–S16) selected from more than 400 L. garvieae isolates were used to induce lysogenic bacteriophages. These strains were treated with 500 ng mL^?1 freshly prepared mitomycin C. A cross‐spotting assay was performed to validate the lysogenic and indicator strains. The lysogenic strains were selected for isolation and concentration of the phages. Phage DNA was digested with EcoRI for biased sinusoidal field gel electrophoresis analysis. Polymerase chain reaction (PCR) was used to detect integrated prophage DNA. Of the 16 representative bacterial genotypes, 12 strains integrated prophages as indicated by the PCR assay, and 10 phages were detected and isolated using two indicator bacterial strains. Analysis of genomic DNA showed that these phages were homologous and named as PLgT‐1. Transmission electron microscopy revealed that the morphology of PLgT‐1 was consistent with the virus family Siphoviridae. PCR analysis of the prophage DNA revealed that all of the S1 genotype strains were lysogenic (30/30), but none of the S16 genotype strains were lysogenic (0/30). This is the first study to investigate lysogenic bacteriophages from L. garvieae.     

Epidemiological study on <Emphasis Type="Italic">Lactococcus garvieae</Emphasis> isolates from fish in Japan

In Japan, Lactococcus garvieae infection has been the main fish disease in aquaculture. Although commercial oral and injectable vaccines have been used to prevent L. garvieae infection in Japan, L. garvieae has been isolated not only from unvaccinated fish but also from vaccinated fish in which immunity induced by vaccination had diminished. In order to obtain epidemiological information on this fish pathogen, we conducted biased sinusoidal field gel electrophoresis (BSFGE) pattern analysis and phage typing of L. garvieae isolates (n = 427) from fish in Japan. These isolates were obtained from 13 different fish species between 1980 and 2007. In the BSFGE analysis, L. garvieae isolates were classified into 17 groups (S1–S17) based on the SmaI digestion patterns and into four groups (A1–A4) based on the ApaI digestion patterns. Phage typing revealed five different phage susceptibility profiles (A–E) in L. garvieae isolates. Since 2005, comparisons of the results of phage typing and BSFGE have indicated the presence of a novel genotype (S16/A4) with phage type E. All the strains belonging to this type showed lincomycin sensitivity.     

Development of a selective and differential medium for capsulated Lactococcus garvieae

A selective and differential medium termed ‘LG agar’ was developed for the isolation and presumptive identification of Lactococcus garvieae that results in black colonies with red halos. In this study, all 14 strains of L. garvieae and only 9 of the 148 strains representing 38 other species were able to grow on the LG agar. The nine viable strains on LG agar plates (including Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Vibrio fluvialis, Vibrio furnissii, Vibrio mimicus and Vibrio salmonicida) were further differentiated from L. garvieae by various colours or colony features. Colonies isolated from the mixing culture and the infected giant sea perch using LG agar plates were all positively identified as L. garvieae by conventional tests and 16S rDNA sequencing. Furthermore, LG agar discriminated capsulated strains of L. garvieae, which were believed to be correlated with pathogens of fish and shellfish, from non‐capsulated ones by colony appearances. The specificity and differentiating ability of LG agar suggest that this medium displays considerable potential for primary isolation and presumptive identification of L. garvieae from pathological and environmental samples.     

Comparison of pulsed‐field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR and biochemical tests to characterize Lactococcus garvieae

Biochemical test, pulsed‐field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC‐PCR) were used to compare 42 strains of Lactococcus garvieae isolated from different regions of Turkey, Italy, France and Spain. Twenty biotypes of L. garvieae were formed based on 54 biochemical tests. ERIC‐PCR of genomic DNA from different L. garvieae strains resulted in amplification of multiple fragments of DNA in sizes ranging between 200 and 5000 bp with various band intensities. After cutting DNA with ApaI restriction enzyme and running on the PFGE, 11–22 resolvable bands ranging from 2 to 194 kb were observed. Turkish isolates were grouped into two clusters, and only A58 (Italy) strain was connected with Turkish isolates. Similarities between Turkish, Spanish, Italian and French isolates were <50% except 216‐6 Rize strain. In Turkey, first lactococcosis occurred in Mugla, and then, it has been spread all over the country. Based on ERIC‐PCR, Spanish and Italian strains of L. garvieae were related to Mugla strains. Therefore, after comparing PFGE profiles, ERIC‐PCR profiles and phenotypic characteristics of 42 strains of L. garvieae, there were no relationships found between these three typing methods. PFGE method was more discriminative than the other methods.     

Detection and quantification of Aeromonas salmonicida in fish tissue by real‐time PCR

Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real‐time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real‐time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real‐time PCR compared to 30% by a culture approach). Also, no real‐time PCR‐negative samples were found positive by culturing. A. salmonicida was detected by real‐time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real‐time PCR showed the presence of the bacterium in all examined organs (1–482 genomic units mg^?1). With a limit of detection of 40 target copies (1–2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real‐time PCR assay provides a sensitive tool for the detection of A. salmonicida.     

Identification of Yersinia ruckeri from diseased salmonid fish by Fourier transform infrared spectroscopy

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), which mainly affects salmonid fish. Isolates of Y. ruckeri from diseased salmonid fish were obtained over a 6‐year period from eight fish farms in the State of Baden‐Württemberg, Southwest Germany. The strains were characterized by biochemical methods and Fourier transform infrared spectroscopy (FT‐IR) combined with artificial neural network analysis. These methods were complemented by 16S rDNA sequencing for several isolates. The set of strains from these fish farms included sorbitol‐positive, gelatinase‐positive and non‐motile Y. ruckeri. These variants were differentiated with an advanced FT‐IR module, which is part of a higher‐ranking method including more than 200 well‐defined Yersinia strains against a background of more than 1000 other Gram‐negative isolates. Validation of the newly constructed method yielded 97.4% of Y. ruckeri identified correctly on the species level. Thus, the FT‐IR analysis enables distinction of all Y. ruckeri from other Yersinia species (e.g. fish‐borne Y. enterocolitica) and other Enterobacteriaceae typically misidentified because of similar biochemical reaction profiles, especially Hafnia alvei. The differentiation of sorbitol‐positive variants of Y. ruckeri using FT‐IR was demonstrated.     

Development and validation of a real‐time PCR assay for the detection of Aeromonas salmonicida

A real‐time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10⁴ ± 1 × 10⁴ CFU g^?1 (without enrichment) and 40 ± 10 CFU g^?1 (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real‐time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real‐time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real‐time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.     

Studies on the inactivation of selected viral and bacterial fish pathogens at high pH for waste disposal purposes

This study investigated the use of alkaline hydrolysis at ambient temperature for inactivation of selected fish pathogens in fish tissues under conditions approximating those that are likely to be found in the aquaculture industry. Infectious salmon anaemia virus (ISAV) and Lactococcus garvieae have been determined in a previous study to be the most resistant virus and bacteria to pH 12 from a wide range of viruses and bacteria tested. They were spiked at high titres into fish extracts that were then treated with 1 m sodium hydroxide (NaOH). Viable L. garvieae was not detected in the treated fish extract after 1 h, and ISAV was not detected after 24‐h exposure. Field mortalities of Atlantic salmon, Salmo salar L., caused by infectious pancreatic necrosis virus were treated by alkaline hydrolysis at ambient temperature. The macerated fish mortalities contained a high titre of virus (3.38 × 10⁸ TCID₅₀ g^?1) that was reduced to approximately 2.2 × 10³ TCID₅₀ g^?1 after 24‐h exposure to NaOH, and virus was not detected after exposure for 48 h. The results suggest that alkaline hydrolysis at ambient temperature has potential as a biosecure treatment method for fish by‐products containing fish pathogens.     

Streptococcosis,Lactococcosis and Enterococcosis are potential threats facing cultured Nile tilapia (Oreochomis niloticus) production

Streptococcosis, lactococcosis and enterococcosis are among the most important bacterial diseases affecting tilapia farms in Kafr Elsheikh governorate, Egypt. A number of clinically diseased fish were collected and submitted to our laboratory during disease outbreak in 2018. They were characterized by nervous swimming behaviour, skin darkness, exophthalmia, ocular opacity and haemorrhages. Necropsy findings were splenomegaly, congestive hepatomegaly, liquefied brain and enteritis. The phenotypic and molecular characterizations of the bacterial strains isolated from naturally infected fish identified three genera of Gram‐positive cocci: Streptococcus agalactiae, Enterococcus faecalis and Lactococcus garvieae. Infectivity trials were conducted in four groups of Nile tilapia inoculated with S. agalactiae Egy‐1, E. faecalis Egy‐1 and L. garvieae Egy‐1 strains and saline. Mortalities, clinical signs and pathological findings were recorded daily 14 days post infection. Experimentally infected tilapia showed similar clinical signs, postmortem lesions, but varied in the severity and experienced high mortalities up to 70% in case of S. agalactiae and L. garvieae infections and 30% in case of E. faecalis infection. Pathological examination of infected tissue sections stained with modified Brown–Brenn and immunohistochemistry revealed an important direct correlation between the distribution of each bacterial isolate and the lesions developed in different organs. Furthermore, the isolates were subjected to profiling against 11 antibiotics, and they showed resistance against several types of antibiotics, which implicate potential risk to human health and emphasize the urgent need for alternate bio‐control strategies to prevent the diseases and the problem of multidrug resistance in aquatic environment.     

Identification and characterization of lactic acid bacteria isolated from rainbow trout (Oncorhynchus mykiss,Walbaum 1792), with inhibitory activity against Vagococcus salmoninarum and Lactococcus garvieae

In this study, a total of 98 lactic acid bacteria isolated from rainbow trout intestines were screened for their probiotic properties. The isolates were tested for their ability to inhibit growth of Vagococcus salmoninarum and Lactococcus garvieae. Based on in vitro antagonism, 10 isolates were selected and evaluated pathogenicity in rainbow trout. Isolates were further investigated for hydrophobicity, bile salts and acid tolerance. These isolates were able to survive low pH and high bile concentrations and showed good adherence characteristics. Isolates were characterized phenotypically, and then, 16S rRNA gene sequence analysis was used for confirmation. Selected strains were administered orally at 10⁸ cfu/g feed, and fish were challenged with V. salmoninarum and L. garvieae. The fish fed with lactic acid bacteria supplemented diets did not improve protection against V. salmoninarum. However, administration of Lactococcus lactis subsp. lactis M17 2‐2 and Lactobacillus sakei 2‐3 resulted in a significant reduction in mortality due to L. garvieae when compared to the control fish. RPS values were calculated as 80 and 53% in fish fed with L. sakei 2‐3 and L. lactis subsp. lactis M17 2‐2, respectively. Our results suggest that these strains could provide an alternative for lactococcosis control in aquaculture.     

Identification and characterization of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae

A study was conducted to evaluate the probiotic properties of endogenous rainbow trout microbiota against pathogenic Lactococcus garvieae. A total of 335 bacterial strains were isolated from rainbow trout and screened for antagonistic activity against L. garvieae using an agar spot assay. Antagonistic strains were grouped by PCR amplification of repetitive bacterial DNA elements (rep‐PCR) and identified by 16S rRNA gene sequence analysis. The results revealed that the antagonistic strains belonged to the genera Lactobacillus, Lactococcus and Leuconostoc. Further probiotic characteristics, such as specific growth rate, doubling time, resistance to biological barriers, antibiotic resistance, hydrophobicity and production of antimicrobial substances, were also studied. These strains were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The antagonistic efficacy was maintained after sterile filtration and was sensitive to proteinase K, indicating that proteinaceous extracellular inhibitory compounds were at least partially responsible for pathogen antagonism. Based on these results, these strains should be further studied to explore their probiotic effects in challenge experiments in vivo. This study shows clear evidence that the indigenous trout‐associated microbiota may provide a defensive barrier against L. garvieae.     

Development of real‐time PCR for detection and quantitation of Streptococcus parauberis

Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real‐time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real‐time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra‐ and interassay coefficient of variation (CV) values ranged from 0.42–1.95%, demonstrating that the assay has good reproducibility. There was not any cross‐reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real‐time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real‐time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples.     

Identification and typing of Vagococcus salmoninarum using genomic and proteomic techniques

This study reports on the characterization of Vagococcus salmoninarum using phenotypic, serological, antigenic, genetic and proteomic methods. All strains of V. salmoninarum were resistant to most of the antimicrobials tested, and only 10% of strains were sensitive to florfenicol. Serological analysis demonstrated a high antigenic homogeneity within the species. No cross‐reaction was detected with other fish pathogenic species causing streptococcosis (Lactococcus garvieae, Streptococcus parauberis, Streptococcus iniae, Streptococcus agalactiae, Carnobacterium maltaromaticum) using serum against V. salmoninarum CECT 5810. Electrophoretic analysis of cell surface proteins and immunoblot supported the antigenic homogeneity within V. salmoninarum strains. Moreover, limited diversity was detected using genomic (RAPD, ERIC‐PCR and REP‐PCR) and MALDI‐TOF‐MS analyses. The phenotypic, genomic and proteomic methods tested allowed the rapid differentiation of V. salmoninarum from the other species causing streptococcosis. However, MALDI‐TOF‐MS is the most promising method for typing and characterization of V. salmoninarum.     

Detection of infectious pancreatic necrosis virus (IPNV) from asymptomatic redbanded seabream,Pagrus auriga Valenciennes,and common seabream,Pagrus pagrus (L.), using a non‐destructive procedure

A non‐destructive procedure based on nested RT‐PCR and dot‐blot hybridization has been developed for the detection of asymptomatic IPNV‐carrier fish. The pair of primers designed for RT‐PCR amplified a 599‐bp fragment of the pVP2 region within the polyprotein gene, resulting in the detection of IPNV genotype III.1. The use of a nested RT‐PCR allowed the amplification of IPNV genotypes III.1 and I.2. In addition, a 191‐bp probe was designed for hybridization studies used in combination with the nested RT‐PCR. The application of the nested RT‐PCR to analyse blood samples from asymptomatic redbanded seabream, Pagrus auriga, and common seabream, P. pagrus, specimens showed a 53.1% and 77.8% prevalence of IPNV‐carriers, respectively. The combination of nested RT‐PCR and dot‐blot hybridization increased the detection rates up to 100% for redbanded seabream and 94.4% for common seabream. Therefore, the protocol described in this study is highly sensitive and specific for the detection of IPNV in asymptomatic carrier fish, and, in addition, the results demonstrate the carrier state in two newly cultured sparid species in southern Spain.     

Rapid and sensitive detection of Vibrio harveyi by loop‐mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene

Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 10² CFU mL^?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 10³ CFU g^?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii.     

Development of a real‐time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single‐step, high‐sensitivity real‐time PCR assay for simultaneous detection and quantification of P. damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory‐generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non‐target bacterial species proved the assay was able to discriminate Phdd‐Phdp subspecies from diverse hosts/geographical origins and between non‐target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp‐Phdd. This is the first time that a molecular method for P. damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease.     

Pre‐challenge and post‐challenge haemato‐immunological changes in Oreochromis niloticus (Linnaeus, 1758) fed argan oil against Lactococcus garvieae

The present study investigated the effects of argan oil, obtained from Argania spinosa, on pre‐ and post‐challenge immuno‐haematological and biochemical responses of Nile tilapia, Oreochromis niloticus. For this purpose, the fish were fed diets containing 0, 0.5%, 1% or 2% argan oil for 45 days. Following 45 days of feeding, fish were challenged with Lactococcus garvieae and mortality was recorded for 15 days. During the pre‐challenge period, significantly higher respiratory burst activity, total white blood cell (WBC), serum lysozyme activity and myeloperoxidase activity were determined in the argan oil‐fed groups. The serum glucose and cholesterol levels decreased whilst total protein and albumin did not change in the groups fed with argan oil‐supplemented diets. After challenge with Lactococcus garvieae, the percentage survival (%) was found to be the highest in the 1% and 2% argan oil‐supplemented feeding groups. Also, there was a significant increase in weight gain, specific growth rate and feed conversion ratio in those fish fed argan oil. The results of this study indicated that after the supplementation of fish diets with argan oil, especially at 1% and 2% concentrations, the immunological, haematological and biochemical values remained similar in both the pre‐ and post‐challenge periods and the immune response against L. garvieae in Nile tilapia was modulated.     

Effect of oregano (Origanum onites L.) essential oil on growth,lysozyme and antioxidant activity and resistance against Lactococcus garvieae in rainbow trout,Oncorhynchus mykiss (Walbaum)

The aim of this study was to investigate the effects of different levels of Origanum onites L. essential oil as feed additives on the growth performance, antioxidant activity and disease resistance of rainbow trout. Fish (26.05 ± 0.15 g) were fed the experimental diets supplemented with four different concentrations (0.125, 1.5, 2.5 and 3.0 mL kg^?1) of O. onites essential oil for 90 days. Fish fed diets containing essential oil of O. onites had significantly higher final weight than the control group. Feed conversion ratio in fish fed diets containing 1.5 and 3.0 mL kg^?1 essential oil of O. onites was improved than other treatments (P < 0.05). The lowest feed conversion efficiency ratio was recorded in the 0.125 mL kg^?1 group of O. onites. Antioxidant status of fish was assayed for levels of plasma superoxide dismutase (SOD) and plasma catalase (CAT) activity. Lysozyme activity in plasma was significantly higher in fish fed diet containing 3.0 mL kg^?1 essential oil of O. onites (P < 0.05). After 8 weeks of feeding, fish were challenged with Lactococcus garvieae and cumulative mortality was recorded over 15 days. Dietary administration of 0.125, 1.5 and 2.5 mL kg^?1 O. onites significantly reduced fish mortality (P < 0.05). The 3.0 mL kg^?1 diet showed no mortality after challenged with L. garvieae. These results suggested that the essential oil of O. onites could be applied as growth promoter and also improved disease resistance when added to rainbow trout feed.