Aeromonas hydrophila-related septicemia in the Nile tilapia Oreochromis niloticus

From diseased wild and cultured Oreochromis niloticus in Lower Egypt, 17 Aeromonas hydrophila isolates were recovered. The mortality was between 10% and 70% in among cultured fish. The course of the disease ran in an acute manner. For cultured fish, the disease outbreaks were found mainly in winter and for the wild Nile fish, mortalities were observed in late spring and summer. Additionally wild fish were affected with ectoparasites. The LD50 values of the isolates ranged between 10(3) and 10(7). Isolates of high virulence were resistant to 1 hr boiling and to the bactericidal effect of fresh normal guinea pig serum. Moreover, they did not agglutinate in acriflavin. Only the virulent isolates could agglutinate tilapia erythrocytes. The above effects were reversed for avirulent isolates while moderately virulent isolates showed no consistency in their reactions. Tube agglutination test using O and WC antisera prepared against 6 isolates versus O and WC antigens of 17 isolates indicated an antigenic heterogenicity of different isolates. While some isolates were identical, 4 antigens out of 17 did not react with any of the sera.     

Purification and localization of nitric oxide synthases from hybrid tilapia (Nile tilapia x Mozambique tilapia)

The aims of this study were to purify and localize the nitric oxide synthases (NOSs) from hybrid tilapia (Nile tilapia Oreochromis niloticus x Mozambique tilapia O. mossambicus). The purification procedures involved affinity chromatography with a 2', 5'-ADP-agarose 4B column and ion exchange with a diethylaminoethanol Bio-Gel A column. The results from gel filtration assays showed that the molecular weights of neuronal NOS (nNOS) and inducible NOS (iNOS) were 178 and 120 kDa, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that there were three bands with molecular weights of 89, 47, and 29 kDa from the purified nNOS. However, only one band, with a molecular weight of 120 kDa, appeared on the gel from the purified iNOS. Hybrid tilapia nNOS was a dimer structure, while iNOS appeared to be a monomer structure. Moreover, our results revealed that the activities of nNOS and iNOS were significantly higher after the addition of Ca+2 or Mg+2 ions individually. However, when L-arginine and NADPH were present, the addition of 1 mM of either ion did not further increase the activity. The chemical L-N(G)-methyl-L-arginine could inhibit the activities of the purified NOSs with or without L-arginine. Western blot analyses showed only an 89-kDa immunoreactive band from the extracts of cerebrum; however, we did not find the specific bands in other tissues, such as gill, intestine, liver, spleen, and anterior kidney. We found another 120-kDa immunoreactive protein band with the rabbit antirat iNOS serum against iNOS from the extracts of anterior kidney and spleen. The results of immunohistochemistry with the rabbit antihuman nNOS serum indicated that the nNOS existed in the cerebellum, olfactory bulb, diencephalons, and nerve cell bodies and neuronal fibers of the spinal cord. Interestingly, only macrophages from anterior kidney and spleen showed positive reactions with the rabbit antirat iNOS serum. In the same way, the endothelial NOS (eNOS) located in the heart and epithelial cells of the blood vessels reacted positively with the rabbit antibovine eNOS serum.     

Bath immersion pharmacokinetics of florfenicol in Nile tilapia (Oreochromis niloticus)

Drug administration by immersion can be a preferable method in certain conditions especially for treating small-sized, anorexic, or valuable fish. Pharmacokinetic information regarding bath treatment is considerably lacking in comparison to other common administration routes. The current study aimed to investigate if immersion can be an effective route to administer florfenicol (FF) for treatment in Nile tilapia. Nile tilapia reared at 28°C were immersed with FF solution at concentrations of 50, 100, 200, 500, and 500/200 (3 hr/117 hr) ppm for 120 hr and moved to drug-free freshwater for another 24 hr. The serum FF concentration in 100, 200, and 500/200 ppm groups reached steady-state at 12 hr with concentrations of 2.44, 3.04, and 5.26 µg/ml, respectively, which were about 2% of the bathing concentrations. The target therapeutic levels of 1–4 µg/ml were attained and maintained within 1–12 hr, depending on the immersion concentration and the target MIC. Serum FF reached the target with shorter time at higher bathing concentration. Following the 120-hr bath, the serum FF declined with the first-order half-life of approximately 10 hr. A minimum of 100 ppm FF is required for treatment purpose, and an initial high loading concentration followed by maintenance concentration is a plausible way to reach in vivo therapeutic level in short time. Greater than 99% of the residual FF in the bathing water could be removed within 15 min by 0.05% NaOCl. Our results indicated that bath immersion is a promising potential route for FF administration in Nile tilapia.     

Edwardsiella ictaluri as the causative agent of mortality in cultured Nile tilapia

Edwardsiella ictaluri was consistently isolated from the spleens, livers, and head kidneys of diseased Nile tilapia Oreochromis niloticus from a farm experiencing mortality events in several culture ponds. We describe the first published outbreak of E. ictaluri-induced edwardsiellosis in Nile tilapia. Pure cultures of the isolated bacteria were characterized both biochemically and molecularly. Biochemical analysis was performed using the API-20E and RapID One systems, and antimicrobial susceptibility was determined by the broth microdilution method. Molecular analysis involved sequencing of the 16S rRNA gene, species-specific real-time polymerase chain reaction (PCR), and PCR-mediated genomic fingerprinting (rep-PCR). Pairwise sequence analysis of the 16S rRNA gene identified the case isolates to be a 100% match to E. ictaluri cultured from channel catfish in the southeastern United States. However, rep-PCR analysis identified the case isolates to be genetically different from representative strains isolated from disease outbreaks in cultured channel catfish in Mississippi. Infectivity challenges (intraperitoneal injection and immersion) demonstrated that a representative E. ictaluri strain isolated from tilapia was pathogenic to naive tilapia, reproducing clinical signs and mortality, thereby establishing Koch's postulates.     

黄霉素对罗非鱼肠道菌群和消化机能的影响

本试验研究了黄霉素对尼罗罗非鱼生长、肠道菌群和消化机能的影响。结果表明,与对照组相比,饲料中添加黄霉素可使罗非鱼日增重和特定生长率分别提高11.11%(P<0.05)和5.38%(P<0.05);饲料转化效率、蛋白质效率和存活率有提高的趋势,但差异均不显著(P>0.05);肠道细菌数量显著降低(P<0.05);干物质和粗蛋白质消化率分别提高5.14%(P<0.05)和3.88%(P<0.05),肠组织蛋白酶活性显著提高(P<0.05);各肠段绒毛和微绒毛高度有增加的趋势,但差异不显著(P>0.05)。     

罗非鱼源无乳链球菌的分离鉴定及药敏试验

从广东省佛山市2个水产养殖场患病罗非鱼中分离到2株细菌,分别编号为T4SH和G6C,经形态学、培养特性观察、生化鉴定和16S rDNA基因序列分析证实为无乳链球菌。2株分离菌株均为革兰阳性菌,呈β溶血;16S rDNA基因序列与无乳链球菌同源性达99%以上。2株菌均对青霉素类、头孢类、林可胺类、大环内酯类、四环素类、氯霉素类抗生素敏感,对氨基糖苷类不敏感;而对磺胺类、喹诺酮类的敏感性存在差异。     

Identification and expression profiles of multiple genes in Nile tilapia in response to bacterial infections

To understand the molecular mechanisms involved in response of Nile tilapia (Oreochromis niloticus) to bacterial infection, suppression subtractive cDNA hybridization technique was used to identify upregulated genes in the posterior kidney of Nile tilapia at 6h post infection with Aeromonas hydrophila. A total of 31 unique expressed sequence tags (ESTs) were identified from 192 clones of the subtractive cDNA library. Quantitative PCR revealed that nine of the 31 ESTs were significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(5)CFU per fish (≈ 20% mortality). Of the nine upregulated genes, four were also significantly (p<0.05) induced in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(6)CFU per fish (≈ 60% mortality). Of the four genes induced by A. hydrophila at both injection doses, three were also significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with Streptococcus iniae at doses of 10(6) and at 10(5)CFU per fish (≈ 70% and ≈ 30% mortality, respectively). The three genes induced by both bacteria included EST 2A05 (similar to adenylate kinase domain containing protein 1), EST 2G11 (unknown protein, shared similarity with Salmo salar IgH locus B genomic sequence with e value of 0.02), and EST 2H04 (unknown protein). Significant upregulation of these genes in Nile tilapia following bacterial infections suggested that they might play important roles in host response to infections of A. hydrophila and S. iniae.     

Class 1 integrons in Aeromonas hydrophila isolates from farmed Nile tilapia (Oreochromis nilotica)

The aim of this study was to determine antimicrobial resistance of Aeromonas hydrophila isolated from farmed Nile Tilapia. A total of 50 A. hydrophila isolates from clinical cases were screened for the presence of class 1, 2 and 3 integrons and all the strains resistant to enrofloxacin and/or ciprofloxacin (n=19) examined for mutation in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. The intI1 gene was detected in 23 A. hydrophila strains (46%) but no intl2 and intl3 were detected. Among these, 14 isolates (60.8%) carried gene cassettes inserted in variable regions i.e., partial aadA2, aadA2, dfrA1-orfC and dfrA12-aadA2, of which the most common gene cassette array was dfrA12-aadA2 (26.09%). Conjugal transfer of class 1 integrons with resistance gene array was detected. All the A. hydrophila strains resistant to enrofloxacin and/or ciprofloxacin possessed mutations in the QRDRs of gyrA and parC. Only a Ser-83-Ile substitution was identified in GyrA and only a Ser-80-Ile amino change was found in ParC. The data confirms that A. hydrophila from farm-raised Nile Telapia serve as a reservoir for antimicrobial resistance determinants.     

Genetic parameters for different growth scales in GIFT strain of Nile tilapia (Oreochromis niloticus)

Body weight, length, width and depth at two growth stages were observed for a total of 5015 individuals of GIFT strain, along with a pedigree including 5588 individuals from 104 sires and 162 dams was collected. Multivariate animal models and a random regression model were used to genetically analyse absolute and relative growth scales of these growth traits. In absolute growth scale, the observed growth traits had moderate heritabilities ranging from 0.321 to 0.576, while pairwise ratios between body length, width and depth were lowly inherited and maximum heritability was only 0.146 for length/depth. All genetic correlations were above 0.5 between pairwise growth traits and genetic correlation between length/width and length/depth varied between both growth stages. Based on those estimates, selection index of multiple traits of interest can be formulated in future breeding program to improve genetically body weight and morphology of the GIFT strain. In relative growth scale, heritabilities in relative growths of body length, width and depth to body weight were 0.257, 0.412 and 0.066, respectively, while genetic correlations among these allometry scalings were above 0.8. Genetic analysis for joint allometries of body weight to body length, width and depth will contribute to genetically regulate the growth rate between body shape and body weight.     

Immunoproteomic analysis of the antibody response obtained in Nile tilapia following vaccination with a Streptococcus iniae vaccine

Streptococcus iniae is one of the most economically important Gram-positive pathogens in cultured fish species worldwide. The USDA-ARS Aquatic Animal Health Research Unit developed a modified (contains concentrated culture supernatant) S. iniae bacterin that has been demonstrated to be efficacious, and protection is mediated by specific anti-S. iniae antibodies. Although effective, the specific vaccine components important for efficacy are not known. In the present study, an immunoproteomic approach was utilized to identify whole-cell lysate proteins of S. iniae that stimulated specific antibody production in Nile tilapia (Oreochromis niloticus) following vaccination. Groups of tilapia were vaccinated by intraperitoneal injection with the modified S. iniae bacterin or were mock-vaccinated, and at 30 d post-vaccination sera samples were obtained from individual fish. Vaccination of tilapia with the S. iniae vaccine stimulated significantly elevated specific antibody responses against proteins of the bacterium and passive immunization of tilapia with this serum demonstrated the antibodies were highly protective. Whole-cell lysate proteins of S. iniae were separated by 2D-PAGE and were probed with a pooled serum sample from vaccinated tilapia. A total of eleven unique immunogenic proteins were positively identified by mass spectrometry. Based on research conducted on homologous proteins in other Streptococcus spp., antibodies specific for three of the identified proteins, enolase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase, are likely involved in protection from streptococcosis caused by S. iniae.     

添加固相负离子粉对吉富罗非鱼生长性能及免疫功能的影响

试验以(15.90±0.1)g的吉富罗非鱼(Oreochromis niloticus)为试验对象,在等氮(33%)等能(17.2MJ/kg)的基础上,在其日粮中分别添加0、0.5%、1%、2%和3%的固相负离子粉,在循环水养殖系统中进行8周的生长试验。每种饲料设计3个重复,每桶放鱼30尾。结果显示:用添加不同剂量的固相负离子粉饲喂吉富罗非鱼,各组间罗非鱼的生长性能、饲料利用率没有显著差异(P<0.05),同时血清中的SOD、LZM、MDA、ALT和AST组间差异均不显著(P>0.05)。     

A piscirickettsiosis-like syndrome in cultured Nile tilapia in Latin America with Francisella spp. as the pathogenic agent

In 2004, cultured Nile tilapia Oreochromis niloticus in several Latin America farms began to succumb to a disease similar to the piscirickettsiosis-like syndrome previously reported in tilapia in Taiwan and the United States. Mortality increased during 2005; reductions in tilapia biomass ranged from 5% to 80% in individual ponds and averaged 50% overall. All ages of fish have been involved. Clinical signs include lethargy, loss of appetite, petechia, exophthalmia, and abnormal swimming behavior. Gross lesions have included splenomegaly, renomegaly, and numerous white nodules observed in the spleen, kidney, testes, heart, ovaries, and occasionally the liver. A previously unreported black granulomatous lesion was reported in up to 30% of the fillets. Histologically, granulomatous infiltrates were observed in the kidney, spleen, liver, testes, ovary, and choroid gland, and rarely in the brain and heart. A small pleomorphic bacterium was observed in Giemsa-stained blood smears and spleen imprints. The bacterium did not grow on standard microbiological media and has not been isolated in cell culture. We obtained a near-complete 16S ribosomal DNA sequence with high similarity to Francisella spp. sequences previously identified in tilapias Oreochromis spp. (Taiwan), Atlantic cod Gadus morhua (Norway), and three-line grunts Parapristipoma trilineatum (Japan).     

Identification and expression profile of multiple genes in Nile tilapia in response to formalin killed Streptococcus iniae vaccination

Twenty-eight expressed sequence tags (ESTs) were isolated from a Nile tilapia (Oreochromis niloticus) vaccinated vs non-vaccinated subtractive library at 12-h post injection of a formalin killed Streptococcus iniae ARS-98-60 vaccine. The 28 ESTs were classified in terms of their putative functions. Half of the ESTs identified were unknown proteins. Of the remaining half ESTs, 17% have putative functions in protein biosynthesis and 11% have putative functions in immunity, energy production, and signal transduction, respectively. Immunity-related ESTs identified included high density lipoprotein-binding protein vigilin, immunoglobulin heavy chain, and QM-like protein. Quantitative PCR revealed that one EST (cytochrome c oxidase subunit II) was highly upregulated (1825 ± 336 fold) in vaccinated fish compared to that in non-vaccinated fish. Of the remaining 27 ESTs, nine were significantly (P<0.05) upregulated (<20 fold) in vaccinated fish. The nine significantly upregulated genes included five unknown or hypothetical proteins and four known proteins (high density lipoprotein-binding protein vigilin, QM-like protein, ribosomal protein S13, and ribosomal protein L5). The upregulation of these genes induced by killed S. iniae vaccines suggest that they might play important role in Nile tilapia defense against S. iniae infection.     

Determination of florfenicol dose rate in feed for control of mortality in Nile tilapia infected with Streptococcus iniae

A dose titration study was conducted to determine the dosage of florfenicol (FFC) in feed to control Streptococcus iniae-associated mortality in Nile tilapia Oreochromis niloticus. Six tanks were assigned to each of five treatments: (1) not challenged with S. iniae and fed unmedicated feed; (2) challenged with S. iniae by injection and fed unmedicated feed; (3) challenged with S. iniae and given FFC at 5 mg/kg of body weight (bw) in medicated feed; (4) challenged with S. iniae and given 10 mg FFC/kg bw; and (5) challenged with S. iniae and given 15 mg FFC/kg bw. Treatment was initiated the day after inoculation, and feed was administered for 10 d. Cumulative mortality was 0% in the unchallenged, untreated group; 35.8 +/- 4.4% (mean +/- SE) in the challenged, unmedicated group; 19.2 +/- 2.7% in the 5-mg/kg treated group, 12.5 +/- 3.8% in the 10-mg/kg group, and 2.5 +/- 1.1% in the 15-mg/kg group. The cumulative mortality was significantly less in each challenged, FFC-treated group than in the challenged, unmedicated controls (5 mg/ kg: P = 0.0156; 10 mg/kg: P = 0.0007; 15 mg/kg: P < 0.0001). The efficacy of the 10- and 15-mg/kg FFC dosages was studied in a separate dose confirmation study. Fish in all tanks were injected with S. iniae. At 4 h postinoculation, 10 tanks were assigned to each of three feed treatments: (1) unmedicated feed; (2) 10 mg FFC/kg bw; and (3) 15 mg FFC/kg bw. Cumulative mortality was 20.5 +/- 2.0% in the challenged, unmedicated group; 11.0 +/- 2.1% in the 10-mg/kg group; and 5.5 +/- 2.4% in the 15-mg/kg group. Mortality was significantly less in the medicated groups than in the challenged, unmedicated control group (10 mg/kg: P = 0.0270; 15 mg/kg: P = 0.0007). Fish in both studies were necropsied, cultured for bacteria, and examined for gross lesions. The minimum inhibitory concentration of FFC against S. iniae in both studies ranged from 0.5 to 1.0 microg/mL. Florfenicol was palatable, safe, and efficacious for control of Nile tilapia mortality due to S. iniae infection.     

Pharmacokinetic study of enrofloxacin in Nile tilapia (Oreochromis niloticus) after a single oral administration in medicated feed

The objective of this study was to evaluate the disposition kinetics of enrofloxacin (ENR) in the plasma and its distribution in the muscle tissue of Nile tilapia (Oreochromis niloticus) after a single oral dose of 10 mg/kg body weight via medicated feed. The fish were kept at a temperature between 28 and 30 °C. The collection period was between 30 min and 120 h after administration of the drug. The samples were analyzed by high‐performance liquid chromatography with a fluorescence detector (HPLC‐FLD). The ENR was slowly absorbed and eliminated from the plasma (C_max = 1.24 ± 0.37 μg/mL; T_max = 8 h; T_1/2Ke = 19.36 h). ENR was efficiently distributed in the muscle tissue and reached maximum values (2.17 ± 0.74 μg/g) after 8 h. Its metabolite, ciprofloxacin (CIP), was detected and quantified in the plasma (0.004 ± 0.005 μg/mL) and muscle (0.01 ± 0.011 μg/g) for up to 48 h. After oral administration, the mean concentration of ENR in the plasma was well above the minimum inhibitory concentrations (MIC₅₀) for most bacteria already isolated from fish except for Streptococcus spp. This way the dose used in this study allowed for concentrations in the blood to treat the diseases of tilapia.     

Dietary L-arginine supplementation reduces lipid accretion by regulating fatty acid metabolism in Nile tilapia(Oreochromis niloticus)

Background: Excessive white fat accumulation in humans and other animals is associated with the development of multiple metabolic diseases. It is unknown whether dietary L-arginine supplementation reduces lipid deposition in high fat diet-fed Nile tilapia(Oreochromis niloticus).Results: In the present study, we found that dietary supplementation with 1% or 2% arginine decreased the deposition and concentration of fats in the liver;the concentrations of triglycerides, low-density lipoprotein, total cholesterol, and high-density lipoprotein in the serum;and the diameter of adipocytes in intraperitoneal adipose tissue. Compared with the un-supplementation control group, the hepatic activities of alanine aminotransferase,aspartate aminotransferase, and lactate dehydrogenase, and hepatic concentration of malondialdehyde were reduced but these for catalase and superoxide dismutase were enhanced by dietary supplementation with 2% arginine. Arginine supplementation reduced the total amounts of monounsaturated fatty acids, while increasing the total amounts of n-3 and n-6 polyunsaturated fatty acids in the liver. These effects of arginine were associated with reductions in mRNA levels for genes related to lipogenesis(sterol regulatory element-binding protein-1, acetyl-CoA carboxylase α, stearoyl-CoA desaturase, and fatty acid synthase) but increases in mRNA levels for genes involved in fatty acid β-oxidation(carnitine palmitoyltransferase 1α and peroxisome proliferator-activated receptor α). In addition, hepatic mRNA levels for Δ4 fatty acyl desaturase 2 and elongase 5 of very long-chain fatty acids were enhanced by arginine supplementation.Conclusion: These results revealed that dietary L-arginine supplementation to tilapia reduced high fat diet-induced fat deposition and fatty acid composition in the liver by regulating the expression of genes for lipid metabolism.     

Utilization of diets containing graded levels of ethanol production co‐products by Nile tilapia

A feeding trial was performed to investigate inclusion levels of distillers dried grains with solubles (DDGS) as a fishmeal replacement for juvenile Nile tilapia (Oreochromis niloticus). On a dry matter basis, five isocaloric [19.3 ± 0.4 kJ/g (mean ± SE)], isonitrogenous (39.1 ± 0.5% crude protein) diets were formulated to contain 17.5%, 20%, 22.5%, 25%, and 27.5% DDGS and compared against a 0% DDGS, reference diet (gross energy = 14.5 kJ/g; crude protein = 39.8%). The reference diet resulted in significantly higher body weight gain (BWG), food conversion ratio (FCR), and protein efficiency ratio (PER) than experimental diets except that 17.5% DDGS provided similar FCR and PER. The diet containing 27.5% DDGS had significantly lower FCR and PER values than all other diets even though apparent digestibility did not significantly differ among experimental diets. Although DDGS can be incorporated at higher levels, 20% DDGS provided the highest apparent BWG among experimental diets, while 17.5% promoted the best FCR and PER. Fishmeal may be replaced with low levels of fuel‐based DDGS to reduce feeding cost; however, additional supplements should be considered to enhance fish performance.     

Efficacy of QCDCR formulated CpG ODN 2007 in Nile tilapia against Streptococcus iniae and identification of upregulated genes

The potential of using a QCDCR (quilA:cholesterol:dimethyl dioctadecyl ammonium bromide:carbopol:R1005 glycolipid) formulated CpG oligodeoxynucleotide (ODN), ODN 2007, to confer protection in Nile tilapia against Streptococcus iniae infection was evaluated in this study. At two days post treatment, QCDCR formulated ODN 2007 elicited significant (P<0.05) protection to Nile tilapia, with relative percent survival of 63% compared to fish treated by QCDCR alone. To understand the molecular mechanisms involved in the protective immunity elicited by ODN 2007, suppression subtractive cDNA hybridization technique was used to identify upregulated genes induced by ODN 2007. A total of 69 expressed sequence tags (ESTs) were identified from the subtractive cDNA library. Quantitative PCR revealed that 44 ESTs were significantly (P<0.05) upregulated by ODN 2007, including 29 highly (>10-fold) and 15 moderately (<10-fold) upregulated ESTs. Of all ESTs, putative peroxisomal sarcosine oxidase was upregulated the highest. The 69 ESTs only included six genes that had putative functions related to immunity, of which only two (putative glutaredoxin-1 and carboxypeptidase N catalytic chain) were confirmed to be significantly upregulated. Our results suggest that the protection elicited by ODN 2007 is mainly through innate immune responses directly or indirectly related to immunity.