Development and validation of glycoprotein‐based native‐subunit vaccine for fish against Aeromonas hydrophila

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein‐based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA‐specific lectin and size‐exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)₃] and Group IV [with Al(OH)₃ and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.     

Effects of dietary supplementation with probiotic live yeast Debaryomyces hansenii on the immune and antioxidant systems of leopard grouper Mycteroperca rosacea infected with Aeromonas hydrophila

Two trials were conducted to determine the efficacy of fish fed live yeast Debaryomyces hansenii strain CBS 8339 on immune and antioxidant systems in leopard grouper Mycteroperca rosacea infected with Aeromonas hydrophila. Juveniles (12±0.5 g) were fed with a control diet or a D. hansenii‐supplemented diet (10⁶ colony‐forming units per gram) for 5 weeks. The live weight of fish was registered on a weekly basis. After 4 weeks, fish from each treatment were immunocompromised with pathogenic A. hydrophila and further fed for 1 week in order to evaluate the effect on immunological and antioxidant parameters. Generally, the results showed enhanced growth performance in fish fed the diet containing yeast compared with the control. Addition of live yeast had no significant effect on the immunological parameters after 4 weeks of feeding. However, post infection with A. hydrophila fish fed the yeast‐supplemented diet resulted in a significant increase in the levels of plasmatic immunoglobulin M. Superoxide dismutase and catalase (CAT) activities were significantly higher in the yeast group. In this fish, CAT and heat shock protein 70 genes were up‐regulated before and after infection of A. hydrophila. The present study is the first one reporting that yeast (D. hansenii) can enhance immunity and resistance against A. hydrophila.     

Haemorrhagic septicaemia in the hybrid surubim (Pseudoplatystoma corruscans×Pseudoplatystoma fasciatum) caused by Aeromonas hydrophila

Intensive culture of the hybrid surubim (Pseudoplatystoma corruscans × Pseudoplatystoma fasciatum) in Brazil is responsible for the occurrence of diseases and consequent economic losses. However, the causative agents are not well known. The objective of this study was to isolate and to characterize the pathogenic agent responsible for mortalities in cultured surubim and to demonstrate its virulence. Ten fish from a fish farm located in the Mato Grosso do Sul State (Brazil) were collected and 14 haemolytic bacteria characterized as Aeromonas hydrophila were isolated from the kidneys (eight) and brain (six). As an experimental challenge, fish weighing 98.1±23.6 g were injected with 1 mL of saline solution and 2 × 10², 2 × 10⁴, 2 × 10⁶ and 2 × 10⁸ CFU A. hydrophila mL^?1. Fish infected with 2 × 10⁸ CFU showed increased external and internal symptoms and mortality of 50±12.5% after 96 h. Increased A. hydrophila concentration was responsible for a decrease in haematocrit percentage and erythrocyte number, lymphocytes and eosinophils, as well as an increase in monocytes, neutrophils, serum agglutination titre and serum antimicrobial activity. It was concluded that A. hydrophila was responsible for characteristic symptoms of bacterial haemorrhagic septicaemia as well as important haematological and immunological alterations, which led to surubim mortality.     

Inhibitory activity of probiotic Enterococcus faecium MC13 against Aeromonashydrophila confers protection against hemorrhagic septicemia in common carp Cyprinuscarpio

The present study involved the control of Aeromonas hydrophila in Cyprinus carpio by bacteria Enterococcus faecium MC13 isolated from fish Mugil cephalus intestine. Antagonistic effects were confirmed against Aeromonas hydrophila by cross-streaking and the agar spot method. Probiotic effects of the isolate were confirmed by injection and oral administration to check for nonpathogenicity to fish. All the two probiotics were orally administered to fingerlings of Cyprinus carpio for 60 days through feed. The intestinal load of bacteria and NBT assay were analyzed on 7, 15, 30, and 60th day of treatment. Experimental fish were challenged with Aeromonas hydrophila on 30 and 60th day. The neutrophil activity was higher in E. faecium MC13 fed fish on day 60 (1.525 ± 0.379 OD). In respect of relative percentage survival (RPS), the higher protection of 75 and 77.8% was observed in E. faecium MC13 fed fish on 30 and 60th day of challenge with Aeromonas hydrophila This study identified the bacterial probiont MC13 (Enterococcus faecium) effectively controlled the Aeromonas hydrophila infection in Cyprinus carpio.     

Some clinical, microbiological and molecular characteristics of Aeromonas hydrophila isolated from various naturally infected fishes

Heat-stable cytotonic enterotoxin gene (Ast) was detected by polymerase chain reaction (PCR) of twenty isolates of Aeromonas hydrophila isolated from various naturally infected fishes collected from both fresh and brackish water. These fishes were Nile tilapia and meagre, mullet and sea bream, respectively. Antibiotic susceptibility, pathogenic characteristics of these isolates and histopathological alterations of liver from experimentally infected tilapia fish with A. hydrophila which contained Ast gene were investigated. PCR technique for the detection of Ast as specific gene for A. hydrophila genomes showed that 90% of tested A. hydrophila (18/20) contained Ast gene, which is specific for A. hydrophila (SSU).The in vitro susceptibility of 18 strains of A. hydrophila (SSU) to 9 antibiotics was evaluated. Oxytetracycline only was an effective antibiotic for all tested isolates. On contrast, all these isolates were resistant to amoxicillin, ampicillin and penicillin. Pathogenicity assay in this study proved that 33.3% of the tested A. hydrophila (6/18) were pathogenic for tilapia in vitro with various levels of virulence where 2/6 were classified as strongly virulent according to the severity of mortality rate. Microscopically, A. hydrophila toxins apparently cause irreparable systemic damage to liver which leads to death.     

Enhancement of protective immunity in blue gourami,Trichogaster trichopterus (Pallas), against Aeromonas hydrophila and Vibrioanguillarum by A. hydrophila major adhesin

Blue gourami, Trichogaster trichopterus (Pallas), were intraperitoneally immunized with major adhesin, a 43 kDa OMP protein isolated from fish Aeromonas hydrophila, in the presence of Freund's complete adjuvant (FCA). Three weeks later, a booster injection of adhesin without FCA was administered. Control group fish were similarly treated with phosphate‐buffered saline (PBS) and FCA. Results showed that anti‐adhesin serum obtained from fish after booster immunization exhibited very strong ability in agglutinating bacterial cells. Although this antiserum had no bactericidal effect, it could significantly inhibit serologically different strains of A. hydrophila from invading EPC (Epithelioma papillosum of carp) cells in vitro. In addition, the proliferative response of head kidney leucocytes of these immune fish was significantly increased as compared to that of the control. The results also showed that the major adhesin could provide significant protective immunity to fish against the challenge by homologous and heterologous strains of A. hydrophila and one virulent strain of Vibrio anguillarum.     

Alterations in the epidermis of the carp,Labeo rohita (Cyprinidae: Cypriniformes), infected by the bacteria,Aeromonas hydrophila: A scanning electron microscopic,histopathological and immunohistochemical investigation

This study was carried out to comprehend the pathogenicity of the bacteria in the epidermis of Labeo rohita inoculated with Aeromonas hydrophila. Alterations in the histopathology of the epidermis were examined using scanning electron microscopy, light microscopy and the localization of iNOS and caspase 3 + ve cells by means of immunohistochemical methods. Skin samples obtained from infected fish at different intervals 2, 4, 6, 8 and 10 days showed significant changes in the cellular components of the epidermis. Epithelial cells often appeared hypertrophied with fragmented and loosely arranged microridges, and in the process of exfoliation. Mucous goblet cells increased significantly in density. Club cells showed degenerative changes, often with simultaneous confluence of adjacent cells and release of their contents. Increase in density of iNOS and caspase 3 + ve cells indicates inflammatory response and apoptosis. This study could provide valuable information on the pathogenesis of the disease, and disease outbreaks in farmed fish. Further, it could provide useful guidelines for fish farmers to take preventive measures for the control of the disease.     

Assessment of Aquaflor(®) , copper sulphate and potassium permanganate for control of Aeromonas hydrophila and Flavobacterium columnare infection in sunshine bass, Morone chrysops female × Morone saxatilis male

Two experiments were conducted to test the effectiveness of different therapeutants against a mixed infection of Aeromonas hydrophila and Flavobacterium columnare in sunshine bass. Experiment 1 evaluated copper sulphate, florfenicol‐medicated feed and potassium permanganate (KMnO₄) against a natural mixed infection. Experiment 2 further evaluated copper sulphate as a treatment to control an experimental mixed infection. In experiment 1, naturally infected untreated fish had the lowest final survival per cent, at 71%, while florfenicol‐medicated feed at 15 mg kg^?1 body weight for 10 days or copper sulphate at 2.1 mg L^?1 (1% of the total alkalinity) for 24 h produced the highest final survivals, at 90% and 88%, respectively. The final survival of the naturally infected fish administered florfenicol‐medicated feed was significantly different (P < 0.1) from the untreated fish. The survival curves for the florfenicol and the copper sulphate at 2.1 mg L^?1 were significantly improved from the untreated fish. In experiment 2, fish were challenged by waterborne exposure to A. hydrophila and F. columnare and either not treated or treated with copper sulphate at 2.1 mg L^?1. At the end of experiment 2, the per cent survival of the challenged fish treated with copper sulphate (99%) was significantly higher (P < 0.05) than the non‐treated (61%). The results illustrate clear benefit of florfenicol and copper sulphate against a mixed infection of A. hydrophila and F. columnare.     

Does the gastrointestinal tract serve as the infectious route of Aeromonas hydrophila in crucian carp (Carassius carassius)?

Under experimental challenges, the gastrointestinal (GI) tract of fish has been proposed as an infectious route of several pathogenic bacteria. Is this also the case for diseased fishponds? A field research was conducted to verify this hypothesis. A crucian carp (Carassius carassius) reared fishpond with motile Aeromonas septicaemia outbreak was sampled in this study. A total of 62 strains of Aeromonas hydrophila were isolated and identified. The clonal relationship among these strains was determined by sequencing the gyrB gene, ERIC‐PCR, RAPD‐PCR, and the presence of seven virulence genes. Strains with identical genotypes were further confirmed as the same clone by multilocus sequence typing analysis. Experimental infection assays were also conducted in zebrafish (Danio rerio). The results show that the same clone strains identical to those in the blood of diseased fish existed in the intestinal digesta of diseased and uninfected fish. Regardless of their origins, all these strains were highly pathogenic to zebrafish. The result indicates that pathogenic strains of A. hydrophila had existed in the GI tract of fish before the infection occurred. This increases our knowledge on infectious route of A. hydrophila in crucian carp.     

An experimental vaccine against Aeromonas hydrophila can induce protection in rainbow trout,Oncorhynchus mykiss (Walbaum)

A candidate vaccine against Aeromonas hydrophila in rainbow trout, Oncorhynchus mykiss, was developed using a bacterial lysate. To test the strength of protection, A. hydrophila challenge models were compared using injection into both the intraperitoneal (IP) cavity and the dorsal sinus (DS) with selected doses of live bacteria washed in saline or left untreated. Unlike the IP route, injection into the DS with either saline washed or unwashed cells resulted in consistent cumulative mortality and a dose response that could be used to establish a standard challenge having an LD₅₀ of approximately 3 × 10⁷ colony forming units per fish. Survivors of the challenge suffered significantly lower mortality upon re‐challenge than naïve fish, suggesting a high level of acquired resistance was elicited by infection. Passive immunization using serum from hyper‐immunized fish also resulted in significantly reduced mortality indicating protection can be transferred and that some portion of resistance may be antibody mediated. Vaccination of groups of rainbow trout with A. hydrophila lysate resulted in significant protection against a high challenge dose but only when injected along with Freund’s complete adjuvant. At a low challenge dose, mortality in all groups was low, but the bacterial lysate alone appeared to offer some protection.     

Development of real‐time and quantitative QCM immunosensor for the rapid diagnosis of Aeromonas hydrophila infection

Since bacterial infection cause a significant economic loss in fish farms, it is necessary to develop rapid diagnostic tools. Interests on label‐free biosensors have been raised for the rapid detection of aquatic pathogenic bacteria but have not been extensively studied yet. Here we report a quartz crystal microbalance (QCM) immunosensor system for the rapid and simple detection of Aeromonas hydrophila, a pathogen for fish and human, in comparison with a conventional indirect ELISA method. In QCM immunosensor system, an antibody against A. hydrophila was covalently cross‐linked to the gold surface of sensor chip and bacterial attachment was monitored as real‐time frequency shifts within 5 min. The frequency shifts were very positively related to the amounts of bacterial cells between 6.25 and 100 μg corresponding to 6 × 10⁶ to 10⁸ CFU with a high specificity. The QCM immunosensor was also able to detect bacterial cells in fish tissue extract in a dose‐dependent manner. Indirect ELISA also showed the dose‐dependent reaction and the amplified signal may allow a lower detection limit. However, QCM immunosensor system showed a more linear and reliable standard curve with R² value of almost 1 (0.9999). Moreover, detection of the bacteria was much quicker and simpler.     

引起混养塘中异育银鲫和鲢发病死亡的病原及组织病理

混养的异育银鲫和鲢鱼种大批死亡,为明确发病死亡的病原和组织损伤并提供相关的疾病防控措施,进行了病鱼肉眼和显微镜检查、细菌学检测、病毒学检测、组织病理和药敏试验研究。结果发现,除在患病鱼体表偶然发现有少量不会引起充血等症状的杯体虫和车轮虫外,未在体内外发现其他寄生虫和真菌类病原;通过细菌分离、人工回感试验、生理生化特性和16S r RNA基因序列分析,从患病异育银鲫和鲢分离到的致病菌株均为嗜水气单胞菌;根据异育银鲫和鲢病毒性疾病的现状,使用鲤疱疹病毒2型(Cyprinid herpesvirus 2,Cy HV-2)DNA聚合酶基因的特异性引物分别对自然发病的异育银鲫和鲢进行PCR检测,只有异育银鲫检测到Cy HV-2,分别用它们的除菌组织上清液进行人工感染试验,只有异育银鲫出现充血症状和死亡现象;由此得出嗜水气单胞菌是异育银鲫和鲢发病死亡的主要病原,Cy HV-2是异育银鲫混合感染的次要病原。患病鲢与患病异育银鲫呈现出类似的组织病理现象,又有一些各自特有的组织病理表现,单纯细菌感染的鲢轻度病变以细胞颗粒变性为主,坏死细胞以核溶解为主,细菌和病毒混合感染的异育银鲫肝脏轻度病变以细胞滴状玻璃样变的变性为主,坏死组织细胞以核固缩和核碎裂为主,在肾脏和脾脏出现染色质边集于核膜的肿大细胞核,主要组织器官出现从变性到坏死的病理变化过程,最终失去应有的功能而死亡。依据药敏试验结果,建议内服诺氟沙星和氟苯尼考等抗生素防治本病的嗜水气单胞菌感染,混合感染Cy HV-2的异育银鲫可以通过注射Cy HV-2疫苗和生态养殖的方法控制和减少该病毒病感染和发展。     

Improvement of intestinal barrier,immunity, and meat quality in common carp infected by Aeromonas hydrophila using probiotics

The fish pathogen Aeromonas hydrophila causes gastrointestinal tract infections and hemorrhagic septicemia and represents a widespread risk in aquaculture. This study aimed to determine whether compound probiotics could improve the intestinal barrier, immunity, and meat quality of common carp infected by A. hydrophila by feeding them compound probiotics. Carp were assigned to one of four groups: (1) control (no infection, no probiotics); (2) control?+?probiotic; (3) A. hydrophila infected; and (4) the A. hydrophila?+?probiotic group. At the beginning of the experiment, the carp were injected with either saline (0.86%) or A. hydrophila (4.87?×?10⁷ CFU/mL). After 2 weeks of the feeding regime, results suggested that in A. hydrophila infected carp, dietary probiotics regulated the intestinal microflora as evidenced by a reduced abundance of Proteobacteria and increased amounts of Firmicutes and Fusobacteria in the intestine. In addition, dietary probiotics ameliorated both villus swelling and the decrease in villus height induced by A. hydrophila (P?<?0.05). Moreover, probiotics prevented the A. hydrophila-induced decline of Occludin, Claudin-1, and ZO-1 levels in the intestine (P?<?0.05). The addition of probiotics into the feed also increased acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM) activity in the serum (P?<?0.05), in comparison to the A. hydrophila group. Importantly, when compared to A. hydrophila-infected carp, compound probiotics alleviated the decrease in muscle nutrient quality (P?<?0.05). In summary, we show that dietary compound probiotics can prevent the impact of A. hydrophila infection on the intestinal function and disease resistance, while ensuring carp muscle quality.

     

Immunization with recombinant aerolysin and haemolysin protected channel catfish against virulent Aeromonas hydrophila

Aeromonas hydrophila is emerging as one of the major concerns in catfish aquaculture in the Southeastern United States due to recent outbreaks of motile aeromonad septicaemia (MAS) caused by virulent clonal isolates. There is no effective vaccine currently available for the prevention of MAS. In this study, two virulence‐associated proteins of A. hydrophila, aerolysin and haemolysin, were heterologously expressed in Escherichia coli cells. Recombinant aerolysin (rArl) and haemolysin (rHly) were used to immunize catfish. Both rArl‐ and rHly‐induced humoral immune response as evidenced by immunoblotting and cell agglutination; immunized fish had significantly less mortality as compared to control fish upon challenge with virulent A. hydrophila. When a mixture of rArl and rHly was used to immunize the fish, significantly higher relative per cent survival (RPS) was obtained. Sustained RPS of 71–78% were observed at 2–5 weeks post immunization. The results of this study indicated that immunization against aerolysin and haemolysin had significant impact on the establishment of pathogenesis by A. hydrophila, suggesting that these two proteins could serve as general immunogens for future development of recombinant protein vaccines.     

草鱼miR-23a在嗜水气单胞菌感染过程中的调控机制

为探索嗜水气单胞菌感染后草鱼肾脏细胞(Ctenopharyngodon idella kidney,CIK)中miR-23a的调控机制,实验通过实时荧光定量PCR(qPCR)测定了嗜水气单胞菌感染CIK细胞后miR-23a的表达量变化,使用RNAhybrid软件预测miR-23a的靶基因并用双萤光素酶报告基因检测系统进...     

Bioassay‐guided isolation and identification of active compounds from Macleaya microcarpa (Maxim) Fedde against fish pathogenic bacteria

Four alkaloids (Sanguinarine, 6‐Methoxyl‐dihydro‐chelerythrine, Cryptopine and β‐Allocryptopine) were isolated from aerial parts of Macleaya microcarpa (Maxim) Fedde using bioassay‐guided isolation method, and the inhibitory activity of ethanolic extract, various fractions and these four alkaloids against four fish pathogenic bacteria (Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum and Vibrio harveyi) was assessed in vitro using the agar dilution method and the microdilution assay method respectively. A. hydrophila was the most sensitive strain to all the tested compounds. Minimum inhibitory concentration (MIC) values were lower for sanguinarine against all tested Gram‐negative strains than other three alkaloids, with MIC values of 12.5 mg L^?1 for A. hydrophila and 50 mg L^?1 to other pathogenic bacteria. Followed by 6‐methoxyl‐dihydro‐chelerythrine, which showed considerable antibacterial activity with MIC values of 80 mg L^?1 for A. hydrophila, 100 mg L^?1 for V. harveyi, and 125 mg L^?1 for both V. anguillarum and A. salmonicida. Cryptopine and β‐allocryptopine revealed similar inhibitory activity with MIC values of 100 mg L^?1 for A. hydrophila and 200 mg L^?1 for other three bacterial species. These finding provided evidence that extract, as well as isolated compounds from M. microcarpa might be potential sources novel antibacterial agents for the treatment of fish infectious diseases.     

Alternate weekly exchanges of feeding regime affect the diversity of intestinal microbiota and immune status of Nile tilapia Oreochromis niloticus

The intestinal microbiota play an important role in the maintenance of the fish health. In this study, 150 Oreochromis niloticus were divided into five groups (G1–G5) and were subjected to alternate weekly exchange of feeding rate and frequency (3, 3, 1.5, 1.5 and 0%) and (2, 1, 2, 1 and 0 time), respectively, for 8 weeks. Enumeration of total intestinal bacteria revealed that the abundance of Lactobacillus was negatively correlated with those of Aeromonas, Pseudomonas and Edwardsiella. The abundance and proportion of Lactobacillus were affected by the change in feeding frequency. When fish were infected with Aeromonas hydrophila, survival rate significantly decreased in G5 (starved fish) compared with G1 (control group). The pro‐inflammatory cytokines, interleukin (IL)‐1β and tumour necrosis factor‐α, and anti‐inflammatory cytokine IL‐10 as well as serum antibacterial activity and respiratory burst activity were positively correlated with the proportion of Lactobacillus. The alternate weekly exchange of feeding regime did not alter the parametric morphology of the intestine, height and width of intestinal villi, number of goblet cells and width of muscle layer, except in G5. In conclusion, the alternate weekly exchange of feeding regime to starvation (G5), decrease in the feeding frequency to 1 time/day (G3) and decrease in feeding rate to 1.5% (G4) suppressed the immune status of fish, which became vulnerable to bacterial infection. Immune suppression was positively correlated with a decreased proportion of Lactobacillus spp.     

L-半胱氨酸提高嗜水气单胞菌对氟苯尼考的敏感性

抗菌药物的应用能对细菌感染性疾病进行有效地控制和治疗,但水产动物养殖中滥用和误用抗菌药物导致耐药细菌的产生、养殖水环境污染和养殖动物药物残留等一系列问题,应对和解决抗菌药物在水产动物养殖中的滥用问题已经刻不容缓。本研究通过抗菌药物杀菌、组织细菌清除、细菌攻毒、鱼体存活率统计和药物敏感性检测等方法,探究外源L-半胱氨酸添加对氟苯尼考杀菌作用的影响。体外杀菌结果显示,4.00 mmol/LL-半胱氨酸可使20.00～200.00μg/mL氟苯尼考对嗜水气单胞菌的杀菌效率提高1.9～11.1倍,其联合用药的杀菌作用具有剂量和时间依赖效应。组织细菌清除结果显示,相比单独使用氟苯尼考,L-半胱氨酸与氟苯尼考合剂对鱼体肝脏、脾脏和肾脏组织中嗜水气单胞菌的清除能力提高4～16倍。细菌攻毒后,采用注射和口服L-半胱氨酸或/和氟苯尼考进行治疗,结果发现L-半胱氨酸与氟苯尼考合剂可大幅提高黄河鲤对嗜水气单胞菌的抗感染能力。同时,单独添加L-半胱氨酸也能促进组织细菌的清除和鱼体的抗感染能力。细菌MIC测定结果显示,添加1.00～4.00 mmol/L L-半胱氨酸使嗜水气单胞菌对氟苯尼考的敏感性提高2～4倍。由此推测,L-半胱氨酸可能通过增强嗜水气单胞菌对氟苯尼考的敏感性,从而提高抗菌药物对鱼体细菌感染的防治效果。研究表明,L-半胱氨酸作为氟苯尼考的增效剂不仅可提高抗菌药物的防治效果,而且可大幅降低抗菌药物的使用量,对嗜水气单胞菌的防控具有一定的实际应用价值。     

Short-term baths with essential oils of Lippia sidoides,Ocimum gratissimum and Zingiber officinale influence blood parameters and survival of tambaqui (Colossoma macropomum) after infection with Aeromonas hydrophila

This study evaluated the effect of short-term baths with the essential oils (EOs) of Lippia sidoides, Ocimum gratissimum and Zingiber officinale on the survival, haematological, biochemical and histopathological parameters of native fish tambaqui (Colossoma macropomum), challenged with Aeromonas hydrophila. After challenge, the fish infected with A. hydrophila received therapeutic baths of 60 min, for five consecutive days. Treatments were as follows: challenged and not treated; challenged and treated with 10 mg L^?1 of gentamicin; challenged and treated with EO of L. sidoides (2.5 and 5.0 mg L^?1); challenged and treated with EO of O. gratissimum (5.0 and 10.0 mg L^?1); challenged and treated with Z. officinale EO (5.0 and 10.0 mg L^?1). The highest survival rates were 89.5% (fish treated with 5.0 mg L^?1 of O. gratissimum EO) and 75.0% (fish treated with 5.0 mg L^?1 of L. sidoides EO). Reductions in the haematocrit percentage, haemoglobin and erythrocyte number were observed in fish treated with L. sidoides and Z. officinale EOs compared with control fish. Mild to moderate damage to the liver tissue of fish after challenge and EO treatments were observed. It is suggested that baths containing O. gratissimum and L. sidoides EOs consist in a viable treatment to increase survival rates of C. macropomum infected with A. hydrophila.     

Determination of the muscle residue levels and withdrawal time of florfenicol in healthy and experimentally Lactococcus garvieae infected rainbow trout (Oncorhynchus mykiss,Walbaum 1792) grown in the Black Sea water

The aim of this study was to determine, muscle residue level (MRL) and withdrawal time (WT) of florfenicol (FF), in healthy and infected rainbow trout (Oncorhynchus mykiss) with Lactococcus garvieae. Fish were divided into the four groups, Group I (Infected fish fed with FF free diet, n = 10), Group II (Healthy fish fed with FF free diet, n = 10), Group III (Infected fish fed with FF containing diet, n = 80) and Group IV (Healthy fish fed with FF containing diet, n = 80). Fish in Group I and Group III were infected with 1.8×10⁶ cfu/ml Lactococcus garvieae by immersion. FF containing diet was applied fish (10 mg kg^‐1 day^‐1) in Group III and IV for 10 days. FF residues in fish muscles were analysed by validated HPLC method. The limit of detection (LOD) and limit of quantification (LOQ) values of the method were determined to be 0.31 μg/g and 0.64 μg/g, respectively, and the recovery was 92.61%. As a result of the experiments, there was no significant difference between the infected and healthy group in term of MRLs. However, in both groups, it was determined that third‐day values were below MRLs (1,000 μg/kg). Also, the WT values were lower than the 500°C‐day values reported in the EC commission decision in both groups and were determined as 14.5 and 14.12 (water temperature mean 18°C ± 1) per day, respectively.