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.     

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.     

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.     

Lactococcus garvieae outbreaks in Brazilian farms Lactococcosis in Pseudoplatystoma sp. – development of an autogenous vaccine as a control strategy

This study evaluated the control of streptococcosis outbreaks in Brazil, isolated from diseased sorubim and identified as Lactococcus garvieae by genetic sequencing. This report determined the potential for lactococcosis control in sorubim Pseudoplatystoma sp. with two vaccines: an aqueous‐based, whole‐cell inactivated vaccine (bacterin) and an oil‐adjuvanted bacterin. Their efficacy was evaluated at 30 days post‐vaccination (d.p.v.) by challenge with L. garvieae, and the antibody production response at 15, 30 and 60 d.p.v. and the non‐specific immune response were compared amongst treatments. High protection levels (P < 0.05) were achieved with the oil‐adjuvanted vaccine with a relative percentage survival value of 81.7% at 30 d.p.v. Additionally, the oil‐adjuvanted vaccine increased the immunogenicity of the bacterin as indicated by greater agglutination antibody titres from 15 until 60 d.p.v. This is the first report of a positive effect of vaccine administration on the specific immunity of sorubim, and the study showed that a specific antibody plays an important role in sorubim defence against lactococcosis because the innate immune responses were similar in all of the studied animals. These results demonstrated that oil‐adjuvanted vaccine can be an effective alternative for the protection of sorubim from L. garvieae 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.     

Susceptibility of selected freshwater fish species to a UK Lactococcus garvieae isolate

Gram-positive cocci recovered from diseased rainbow trout from a farm in England were characterized by different methods, including pulsed field gel electrophoresis, as virulent Lactococcus garvieae serogroup 2 (pulsotype A1). Groups of rainbow trout were kept at a range of temperatures and injected intraperitoneally (i.p.) with one of the UK isolates, L. garvieae 00021. The 18 °C and 16 °C groups showed 67% and 28% mortality, respectively, by day 27 post-injection. Fish kept at 14 °C or lower were less susceptible (≤3% mortality). Raising the temperature of all groups to 18 °C at day 27 post-injection did not result in recurrence of the disease, even though viable bacteria were recovered from all groups 42 days later. Grayling were highly susceptible, with 65% mortalities when challenged with 200 colony forming unit fish⁻¹ by i.p. injection and 37% mortalities when exposed to effluent water from tanks containing affected rainbow trout. Other fish species tested, Atlantic salmon, brown trout and seven cyprinid species, were less susceptible. Viable L. garvieae was isolated from the internal organs of all species tested at the end of the trials, suggesting that they may pose a threat as possible carriers to susceptible farmed and wild fish.     

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.     

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.     

7种海洋鱼类的生物能量学模式

根据室内流水实验法，定量研究了黄、渤海生态系统7种鱼类能量收支组分，并建立了相应的生物能量学模式。7种海洋鱼类的生物能量学模式豆著不同，并可分为3类：(1)较低代谢和较高生长，如黑鲳、矛尾鳃虎鱼；(2)较高代谢和较低生长，如欧氏六线鱼和黑鲷；(3)代谢和生长均处于中等水平，如真鲷、红鳍东方纯和鲐。其差异原因可能与这7种海洋鱼类的生态习性不同相关。与淡水肉食性鱼类比较，7种海洋鱼类的代谢能明显偏高，表明海洋鱼类属于高代谢消耗、低生长效率型鱼类。     

Preliminary characterization of Lactococcus garvieae bacteriophage isolated from wastewater as a potential agent for biological control of lactococcosis in aquaculture

Lactococcosis, a significant emerging disease of fish caused by Lactococcus garvieae, has become one of the devastating problems due to its serious economic damage in aquaculture. The aim of this study was to isolate and characterize a lytic phage infecting L. garvieae as a potential bioagent for the treatment of lactococcosis. In this regard, one strain of L. garvieae was isolated from diseased rainbow trout, and then, following biochemical and molecular identifications, its specific phage, WWP-1, which was able to destroy L. garvieae cells through the lytic cycle, was isolated from a municipal wastewater sample. Transmission electron microscope revealed that the isolated phage possesses an icosahedral head and a non-contractile short tail, resembled to members of the family Podoviridae. Moreover, phage WWP-1 represented optimal antibacterial activity at temperatures ranging from 15 to 30 °C, suggesting that it could be very effective at rainbow trout rearing temperature. Restriction profile analysis revealed that NdeI can digest WWP-1 genome while EcoRI, EcoRV, and BamHI were incapable of cutting its DNA. According to the in vivo experiment result, WWP-1 could decrease mortality rate of infected rainbow trout in aquaculture. The results suggest that this naturally occurring bacteriophage could be considered as a promising agent to control the disease caused by L. garvieae strains in rainbow trout rearing.     

Polyphasic characterization of Aeromonas salmonicida isolates recovered from salmonid and non‐salmonid fish

Michigan's fisheries rely primarily upon the hatchery propagation of salmonid fish for release in public waters. One limitation on the success of these efforts is the presence of bacterial pathogens, including Aeromonas salmonicida, the causative agent of furunculosis. This study was undertaken to determine the prevalence of A. salmonicida in Michigan fish, as well as to determine whether biochemical or gene sequence variability exists among Michigan isolates. A total of 2202 wild, feral and hatchery‐propagated fish from Michigan were examined for the presence of A. salmonicida. The examined fish included Chinook salmon, Oncorhynchus tshawytscha (Walbaum), coho salmon, O. kisutcha (Walbaum), steelhead trout, O. mykiss (Walbaum), Atlantic salmon, Salmo salar L., brook trout, Salvelinus fontinalis (Mitchill), and yellow perch, Perca flavescens (Mitchill). Among these, 234 fish yielded a brown pigment‐producing bacterium that was presumptively identified as A. salmonicida. Further phenotypic and phylogenetic analyses identified representative isolates as Aeromonas salmonicida subsp. salmonicida and revealed some genetic and biochemical variability. Logistic regression analyses showed that infection prevalence varied according to fish species/strain, year and gender, whereby Chinook salmon and females had the highest infection prevalence. Moreover, this pathogen was found in six fish species from eight sites, demonstrating its widespread nature within Michigan.     

Characterization of bacteriophage HN48 and its protective effects in Nile tilapia Oreochromis niloticus against Streptococcus agalactiae infections

Streptococcus agalactiae is a causative agent responsible for massive mortalities of tilapia that has led to catastrophic losses to tilapia culture globally. Bacteriophages represent a new class of antimicrobials against bacteria. In this study, we characterized the bacteriophage HN48, which formed small and round‐transparent plaques on a double‐layer plate. With a hexagonal head and a long tail, this phage may belong to the Caudovirales according to the International Committee on Taxonomy of Viruses. HN48 was found to have a relatively wide and highly specific host range, to be sensitive to high temperature (60–80°C) and low pH (3–5), and to be relatively stable at alkaline pH (8–10). Intraperitoneal injection with HN48 had no adverse effects on tilapia and effectively inactivated the bacteria in the kidney. Fish that received phage therapy had 60% ± 3.3% survival rates and a delayed mean death time of about 3 days when compared to the control group. To the best of knowledge, this is the first study of tilapia streptococcal phage. Overall, the results indicated that phage HN48 could prevent tilapia from experimental S. agalactiae infection, suggesting it has the potential to control this disease.     

Multiple dietary administrating strategies of water hyacinth (Eichhornia crassipes) on enhancing the immune responses and disease resistance of giant freshwater prawn,Macrobrachium rosenbergii

The different products of Eichhornia crassipes leaves including dried E. crassipes powder (DEP), hot‐water treated E. crassipe (HTE), hot‐water extract of E. crassipe (ECE) and dreg of hot‐water extract of E. crassipe extract (dECE) were produced and incorporated into the diet of the prawn, Macrobrachium rosenbergii, as an immunostimulant. Results showed that prawn fed the HTE‐, ECE‐ and dECE‐containing diets for 4 months had increased total haemocyte count, different haemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, glutathione peroxidase activity especially in HTE and ECE treatments. The phagocytic activity and clearance efficiency against Lactococcus garvieae of prawn fed the HTE‐ and ECE‐containing diets were significantly higher than those of prawn fed the control diet at 2–4 months. The relative percentage survival of prawn fed the DEP‐, HTE‐, ECE‐ and dECE‐containing diets for 4 months following 144 h challenging with L. garvieae were 19.0%, 38.1%, 38.1% and 33.3%. It was concluded that E. crassipes leaves containing an active component which was easily extracted by hot water can enhance innate immunity and resistance against pathogen of M. rosenbergii by dietary long‐term administration, and the administration of HTE in the diet was the best strategy due to the availability and convenience.     

In vitro selection and characterization of putative probiotics isolated from the gut of Acipenser baerii (Brandt, 1869)

To select and characterize potential probiotic bacteria from the gut microbiota of Siberian sturgeon (Acipenser baerii), 129 strains isolated from the hindgut were screened for antagonistic activity against five fish pathogens. Ten isolates showed antagonism towards three or more pathogens. Nine of these isolates were Gram‐positive, belonging to Lactococcus (seven) and Bacillus (two), and a single strain belonging to the Gram‐negative Citrobacter. These inhibitory isolates were identified using genetic, phentotypic and biochemical traits, and further characterized by in vitro tests assessing the adhesion and growth in mucus and resistance to gastric and intestinal fluids. The candidate probiotics were determined to be non‐pathogenic through an in vivo study. Based on these assays, Lactococcus lactis ssp. lactis STG45 and STG81 showed the broadest inhibitory potential, a high viability in simulated gastrointestinal juice and the highest adhesion capacity to mucus. They were therefore selected as the most promising candidate probiotics. This is the first study screening probiotics among the gut microflora of Siberian sturgeon.     

PCR survey for Paramoeba perurans in fauna,environmental samples and fish associated with marine farming sites for Atlantic salmon (Salmo salar L.)

Amoebic gill disease (AGD) caused by the amoeba Paramoeba perurans is an increasing problem in Atlantic salmon aquaculture. In the present PCR survey, the focus was to identify reservoir species or environmental samples where P. perurans could be present throughout the year, regardless of the infection status in farmed Atlantic salmon. A total of 1200 samples were collected at or in the proximity to farming sites with AGD, or with history of AGD, and analysed for the presence of P. perurans. No results supported biofouling organisms, salmon lice, biofilm or sediment to maintain P. perurans. However, during clinical AGD in Atlantic salmon, the amoeba were detected in several samples, including water, biofilm, plankton, several filter feeders and wild fish. It is likely that some of these samples were positive as a result of the continuous exposure through water. Positive wild fish may contribute to the spread of P. perurans. Cleaner fish tested positive for P. perurans when salmon tested negative, indicating that they may withhold the amoeba longer than salmon. The results demonstrate the high infection pressure produced from an AGD‐afflicted Atlantic salmon population and thus the importance of early intervention to reduce infection pressure and horizontal spread of P. perurans within farms.     

Association of a specific major histocompatibility complex class IIβ single nucleotide polymorphism with resistance to lactococcosis in rainbow trout,Oncorhynchus mykiss (Walbaum)

Major histocompatibility complex (MHC) loci encode glycoproteins that bind to foreign peptides and initiate immune responses through their interaction with T cells. MHC class II molecules are heterodimers consisting of α and β chains encoded by extremely variable genes; variation in exon 2 is responsible for the majority of observed polymorphisms, mostly concentrated in the codons specifying the peptide‐binding region. Lactococcus garvieae is the causative agent of lactococcosis, a warm‐water bacterial infection pathogenic for cultured freshwater and marine fish. It causes considerable economic losses, limiting the profitability and development of fish industries in general and the intensive production of rainbow trout, Oncorhynchus mykiss (Walbaum), in particular. The disease is currently controlled with vaccines and antibiotics; however, vaccines have short‐term efficacy, and increasing concerns regarding antibiotic residues have called for alternative strategies. To explore the involvement of the MHC class II β‐1 domain as a candidate gene for resistance to lactococcosis, we exposed 400 rainbow trout to naturally contaminated water. One single nucleotide polymorphism (SNP) and one haplotype were associated with resistance (P < 0.01). These results are promising for using MHC class IIβ as a molecular marker in breeding rainbow trout resistant to lactococcosis.     

Susceptibility of Caligus rogercresseyi collected from the native fish species Eleginops maclovinus (Cuvier) to antiparasitics applied by immersion

A major challenge for Chilean salmon farming is infestation by the ectoparasite Caligus rogercresseyi. In addition, there is evidence that a loss of chemotherapeutic treatment efficacy against important fish pathogens is occurring in salmon farming, including antiparasitic efficacy. Currently, there are known techniques that allow the determination of the susceptibility profile of parasites to antiparasitic treatment. However, there is scarce information about both threshold values and categorization of antiparasitic susceptibility for C. rogercresseyi. Bioassay technique allowed the determination of both mean values and the natural variation of EC50%, which were contrasted with available susceptibility thresholds. Results allowed to determine that parasites from the native fish host, Eleginops maclovinus, are susceptible to azamethiphos, deltamethrin and cypermethrin treatments, showing a high susceptibility profile to antiparasitics.