Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus)

Flavobacterium columnare, causal agent of columnaris disease, is pathogenic to many species of freshwater fish throughout the world. The United States channel catfish (Ictalurus punctatus) aquaculture industry is severely impacted by columnaris disease. The majority of the F. columnare isolates recovered from diseased channel catfish belonged to either genomovars I or II. The objective of the present study was to determine if differences existed in the ability of these genomovars to induce mortality in channel catfish. Single strand conformation polymorphism analysis (SSCP) was used to ascribe the isolates used in this study to the appropriate genomovar. Immersion challenge experiments (15min immersion exposure to approximately 5x10(5) to 1x10(6) CFU/mL) were carried out to assess virulence of genomovar I and II isolates to channel catfish. The results demonstrated that genomovar II (n=4) isolates were significantly (P<0.05) more virulent to channel catfish fry (92-100% mortality) than genomovar I (n=3) isolates (0-46% mortality). In vivo adhesion of the genetically characterized F. columnare also correlated (r2=0.73) to increased mortality in the challenged fry. In fingerling channel catfish, significantly higher mortality (P<0.05) resulted with genomovar II isolates ALM-05-182 and ALG-00-530 as compared to all the genomovar I isolates (n=3). Mortality of genomovar II isolate BGFS-27 with similar to genomovar II isolate (ALG-00-530) and two genomovar I isolates (ALM-05-53 and 140). The results suggest that although both genomovars are present in the aquatic environment, genomovar II appears to be more pathogenic for channel catfish.     

Colorimetric Method of Loop-Mediated Isothermal Amplification with the Pre-Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms

Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP–calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10² F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP–calcein method had 97% homology with the DNA sequence of F. columnare.

Received May 21, 2014; accepted August 10, 2014     

Isolation and Partial Characterization of Extracellular Proteases Produced by Isolates of Flavobacterium columnare Derived from Channel Catfish

Abstract

Proteases of 23 isolates of Flavobacterium columnare derived primarily from channel catfish Ictalurus punctatus raised in the southeastern United States were isolated and partially characterized. The bacterial isolates were divided into two groups according to the apparent molecular masses of proteases after zymographic resolution by nonreducing, nondenaturing sodium dodccyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with gelatin as the protease substrate. The 15 isolates in group 1 had two proteases with apparent molecular masses of 58 and 53.5 kilodaltons (kDa). Eight group-2 isolates produced three proteases with apparent molecular masses of 59.5, 48, and 44.5 kDa. Culture medium had an effect on the amount of protease produced by F. columnare LA 88–173. More protease was produced in a medium with low nutrients and salt (Ordal's medium) than in media with higher concentrations of nutrients or salts (TYES, Hsu-Shotts, modified Shieh's media). No differences were observed in the apparent molecular masses of the two proteases of F. columnare LA 88–173 produced in the various media or with different incubation times. Two proteases with apparent molecular masses of 58 and 53.5 kDa were seen as early as I d after inoculation, and these molecular masses did not change during the 7-d experiment. A sharp increase in protease production occurred during the first 24 h of incubation with minimal increase during the remaining 7 d of the experiment. All 23 isolates of F. columnare degraded the gelatin and casein incorporated into TYES agar medium but only 7 of the 23 isolates degraded elastin.     

Spleen index and mannose-binding lectin levels in four channel catfish families exhibiting different susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri

Edwardsiella ictaluri and Flavobacterium columnare are two bacterial pathogens that affect channel catfish Ictalurus punctatus aquaculture. At the Catfish Genetics Research Unit (U.S. Department of Agriculture, Agricultural Research Service), some progress has been made in selectively breeding for resistance to E. ictaluri; however, the susceptibility of these families to F. columnare is not known. Our objectives were to obtain baseline information on the susceptibility of channel catfish families (maintained as part of the selective breeding program) to E. ictaluri and F. columnare and to determine whether the spleen index and plasma levels of mannose-binding lectin (MBL) are predictive indicators of susceptibility to these pathogens. Four channel catfish families were used: family A was randomly chosen from spawns of fish that were not selectively bred for resistance; families B, C, and D were obtained after selection for resistance to E. ictaluri. All four families were immersion challenged with both bacterial pathogens; the spleen index and plasma MBL levels of unchallenged fish from each family were determined. Mean cumulative percent mortality (CPM) after E. ictaluri challenge ranged from 4% to 33% among families. Families A and B were more susceptible to F. columnare (mean CPM of three independent challenges = 95% and 93%) than families C and D (45% and 48%), demonstrating that there is genetic variation in resistance to F. columnare. Spleen index values and MBL levels were not significantly different, indicating that these metrics are not predictive indicators of F. columnare or E. ictaluri susceptibility in the four tested families. Interestingly, the two families that exhibited the highest CPM after F. columnare challenges had the lowest CPM after E. ictaluri challenge. Further research on larger numbers of families is needed to determine whether there is any genetic correlation between resistance to E. ictaluri and resistance to F. columnare.     

Spleen Index and Mannose-Binding Lectin Levels in Four Channel Catfish Families Exhibiting Different Susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri

Abstract

Edwardsiella ictaluri and Flavobacterium columnare are two bacterial pathogens that affect channel catfish Ictalurus punctatus aquaculture. At the Catfish Genetics Research Unit (U.S. Department of Agriculture, Agricultural Research Service), some progress has been made in selectively breeding for resistance to E. ictaluri; however, the susceptibility of these families to F. columnare is not known. Our objectives were to obtain baseline information on the susceptibility of channel catfish families (maintained as part of the selective breeding program) to E. ictaluri and F. columnare and to determine whether the spleen index and plasma levels of mannose-binding lectin (MBL) are predictive indicators of susceptibility to these pathogens. Four channel catfish families were used: family A was randomly chosen from spawns of fish that were not selectively bred for resistance; families B, C, and D were obtained after selection for resistance to E. ictaluri. All four families were immersion challenged with both bacterial pathogens; the spleen index and plasma MBL levels of unchallenged fish from each family were determined. Mean cumulative percent mortality (CPM) after E. ictaluri challenge ranged from 4% to 33% among families. Families A and B were more susceptible to F. columnare (mean CPM of three independent challenges = 95% and 93%) than families C and D (45% and 48%), demonstrating that there is genetic variation in resistance to F. columnare. Spleen index values and MBL levels were not significantly different, indicating that these metrics are not predictive indicators of F. columnare or E. ictaluri susceptibility in the four tested families. Interestingly, the two families that exhibited the highest CPM after F. columnare challenges had the lowest CPM after E. ictaluri challenge. Further research on larger numbers of families is needed to determine whether there is any genetic correlation between resistance to E. ictaluri and resistance to F. columnare.

Received November 18, 2011; accepted February 23, 2012     

Development of Similar Broth Microdilution Methods to Determine the Antimicrobial Susceptibility of Flavobacterium columnare and F. psychrophilum

Flavobacterium columnare and F. psychrophilum are major fish pathogens that cause diseases that may require antimicrobial therapy. Choice of appropriate treatment is dependent upon determining the antimicrobial susceptibility of isolates. Therefore we optimized methods for broth microdilution testing of F. columnare and F. psychrophilum to facilitate standardizing an antimicrobial susceptibility test. We developed adaptations to make reproducible broth inoculums and confirmed the proper incubation time and media composition. We tested the stability of potential quality-control bacteria and compared test results between different operators. Log phase occurred at 48 h for F. columnare and 72–96 h for F. psychrophilum, confirming the test should be incubated at 28°C for approximately 48 h and at 18°C for approximately 96 h, respectively. The most consistent susceptibility results were achieved with plain, 4-g/L, dilute Mueller–Hinton broth supplemented with dilute calcium and magnesium. Supplementing the broth with horse serum did not improve growth. The quality-control strains, Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658, yielded stable minimal inhibitory concentrations (MIC) against all seven antimicrobials tested after 30 passes at 28°C and 15 passes at 18°C. In comparison tests, most MICs of the isolates agreed 100% within one drug dilution for ampicillin, florfenicol, and oxytetracycline. The agreement was lower with the ormetoprim–sulfdimethoxine combination, but there was at least 75% agreement for all but one isolate. These experiments have provided methods to help standardize antimicrobial susceptibility testing of these nutritionally fastidious aquatic bacteria.

Received June 24, 2015; accepted October 2, 2015.     

Antagonistic effect of indigenous skin bacteria of brook charr (Salvelinus fontinalis) against Flavobacterium columnare and F. psychrophilum

Industrial fish production exposes fish to potentially stressful conditions, which in turn may induce infections by opportunistic pathogens. Probiotics appear to be a promising way to prevent opportunistic infections in aquaculture. In this study, we tested the inhibitory potential of endogenous bacterial communities found in the mucus of brook charr (Salvelinus fontinalis) against two major pathogens Flavobacterium columnare and Flavobacterium psychrophilum. Nine bacterial strains were isolated from brook charr skin mucus and tested for potential antagonistic activity. Results from both agar diffusion assays and broth co-culture assays showed the presence of antagonism. We identified seven bacterial strains, collected from unstressed fish, which exerted strong antagonism against F. psychrophilum and/or F. columnare. These strains were mixed and used to treat columnaris disease in an in vivo experiment in which four distinct fish families were tested. This treatment resulted in a decrease of mortality (54-86%) across fish families indicating that candidates from the host microbiota are potentially suitable for probiotic development. This would allow for the efficient (ability to adhere and colonize the host mucus) and durable management (antagonistic effect against pathogens which would be harmless for the host and safe for its environment) of opportunistic diseases in aquaculture.     

The association of Flavobacterium columnare strains of high and low virulence with gill tissue of black mollies (Poecilia sphenops).

The ability of a high virulence strain (AJS 1) and a low virulence strain (AJS 4) of Flavobacterium columnare (Flexibacter columnaris) to attach to the gills of black mollies (Poecilia sphenops) was investigated. For that purpose, two groups of 25 black mollies each were immersed in a bath containing 10(6) CFU/ml of F. columnare AJS 1 or AJS 4. At regular intervals from 1 to 12 h after the contact infection, fish were sacrificed and gills, skin, spleen and heart were sampled for bacteriology. Samples of the gills were taken for immunohistochemical and electron microscopic examination. Bacteriological examination proved that the number of gill-associated F. columnare was higher for AJS 1 than for AJS 4. Strain AJS 1 was isolated from the heart and spleen of 6 and 1 of the 16 examined animals, respectively. Strain AJS 4 was not isolated from the internal organs of any fish. When examined immunohistochemically, strain AJS 1 was found closely associated with gill epithelium whereas this was not the case for strain AJS 4. The adherence of bacteria to the gill tissue challenged with the virulent strain AJS 1 was also clearly demonstrated using scanning and transmission electron microscopy. These results indicate that adhesion of F. columnare to the gill tissue constitutes an important step in pathogenesis.     

Determination of Phylogenetic Relationships of Flavobacterium psychrophilum (Flexibacter psychrophilus), Flavobacterium columnare (Flexibacter columnaris), and Flexibacter maritimus by Sequence Analysis of 16S Ribosomal RNA Genes Amplified by Polymerase Chain Reaction

Abstract

The nearly complete small subunit 16S ribosomal RNA (rRNA) gene sequence amplified by polymerase chain reaction was determined for Flavobacterium psychrophilum (formerly Flexibacter psychrophilus) by using automated nucleotide sequencing. The sequence was found to be 1,465 base pairs (bp) in length, a size consistent with previously determined sequences for 14 other bacterial species from various taxa, including the yellow-pigmented bacteria. Sequence signatures confirmed that this organism was a member of the bacterial division Bacteroides–Flavobacterium. Parsimonious and additive phylogenetic trees were constructed with homology, and pairwise evolutionary distances were used to estimate phylogenetic relationships. Data show that F. psychrophilum, F. columnare, and Flexibacter maritimus are closely related, have a common descent, and represent a distinct group within the division Bacteroides–Flavobacterium. This group also included other organisms from the genera Flavobacterium and Cytophaga. Further, Flavobacterium aquatile, the type species for the genus Flavobacterium, was also determined to be a member of this “Flavobacterium–Cytophaga–Flexibacter subcomplex.” This supports previous assertions that the type strain of Flavobacterium should be changed to a more representative species such as Flavobacterium breve.     

活体电穿孔方法提高外源GRF基因在小鼠肌肉组织中的表达

向小鼠后肢小腿肌注pGRF表达质粒并加以电穿孔条件,注射剂量分别为5,10,50μg,于注射前及注射后10,20,30d测体质量,并采血分离血清测血清GRF水平。结果显示,5μgpGRF质粒电穿孔组注射后10dGRF分别比对照组、5μgpGRF质粒组升高44.73%(P0.05),12.86%(P0.05);血清中GRF水平升高。30d累积增重,5μgpGRF质粒电穿孔组分别比对照组、5μgpGRF质粒组高11.46%,6.75%(P0.05);10μgpGRF质粒组分别比对照组和10μgpGRF质粒电穿孔组高19.52%(P0.05),19.42%(P0.05)。50μgpGRF质粒组45d累积增重分别比对照组、pGRF质粒电穿孔组高14.00%,12.00%(P0.05)。结果表明,电穿孔处理可提高GRF基因在小鼠肌肉中的表达。     

RNAi suppression of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) mediated differentially expressed genes involved in Toll-like receptor signaling pathway and caused increased susceptibility to Flavobacterium columnare

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare.     

抗柱状黄杆菌多克隆抗体的制备及其免疫学特性鉴定

为制备抗柱状黄杆菌多克隆抗体,本研究利用颗粒性抗原免疫新西兰白兔,收集的抗血清通过辛酸-硫酸铵法纯化,采用间接ELISA法检测纯化后多克隆抗体的效价和交叉反应性。结果显示,制备的多克隆抗体蛋白质浓度为29.28 mg/mL,效价在1:6.4×10⁴以上,与迟钝爱德华氏菌、大肠杆菌、嗜水气单胞菌、鳗弧菌、溶藻弧菌、副溶血弧菌及哈维氏弧菌等水生动物致病菌均无交叉反应。本研究成功建立了抗柱状黄杆菌多克隆抗体的制备方法,可用于柱状黄杆菌的快速检测。     

Effects of ewe breed and management system on efficiency of lamb production: I. Ewe productivity

Performance of 1/2-Suffolk, 1/2-Rambouillet (Western) and 1/2-Suffolk, 1/4-Rambouillet, 1/4-Finnsheep (1/4-Finn) ewes was compared in three different lamb production systems over 3 yr. System 1 (56 ewes) involved late fall lambing over 84 d. System 2 (51 ewes) involved January and February lambing for 60 d. System 3 (47 ewes) involved March and April lambing for 45 d. Pregnancy rates for yearling ewes were lower in System 1 in yr 1 (50.7% vs 87.4% for Systems 2 and 3) but differed little among systems for older ewes or in remaining years. Average pregnancy rates for 2-yr-old and older ewes were 89.5, 94.0 and 85.7% for Systems 1, 2 and 3, respectively. When the pregnancy rate was adjusted to a 45-d lambing season, means for older ewes were 78.6, 89.5 and 85.7% for System 1, 2 and 3, respectively. Ewe breeds did not differ in their pregnancy rates. Prolificacy (lambs born per ewe lambing) was higher for 1/4-Finn ewes (1.83 +/- .06 vs 1.55 +/- .07) and was higher in System 3 (1.86 +/- .06) than in Systems 1 (1.60 +/- .07) or 2 (1.63 +/- .05). Body weight at breeding in postyearling ewes was less in System 3 (64.3 kg); than in Systems 1 or 2 (average of 73.1 kg). Breeds did not differ in weight at 1 or 2 yr of age, but Western ewes were 2.1 +/- 1.1 kg heavier as 3-yr-olds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survey of restriction-modification systems and transformation in Mannheimia haemolytica and Pasteurella trehalosi

A significant obstacle to molecular studies of Mannheimia (Pasteurella) haemolytica, has been its resistance to genetic transformation. The lack of competence of many M. haemolytica strains has been attributed to the presence of restriction modification systems. In this study, representative strains of 12 M. haemolytica serotypes and four Pasteurella trehalosi serotypes were successfully transformed by electroporation using a recombinant vector derived from the native M. haemolytica A1 serotype plasmid pNSF2176. Transformation was achieved despite PCR-based evidence for the presence of genes encoding a type I restriction enzyme, phaI, and a type II restriction enzyme hsdM, in each of the M. haemolytica strains.     

槲皮素的生物学效应及应用前景

<正>槲皮素(Quercetin)是一种天然的黄酮类化合物,存在于多种植物的花、叶、果实中,具有抗炎、抗氧化、清除氧自由基、抗粘附、抗血栓、抗病毒、抗肿瘤、降血糖、降血脂、降低血压以及免疫调节作用[1,2]。     

鸭Ⅰ型肝炎病毒VP1基因片段的克隆与表达载体的构建

为研究鸭肝炎病毒VP1基因在大肠杆菌中的表达情况,根据鸭肝炎病毒核苷酸序列设计1对外引物和1对含有EcoRⅠ和SalⅠ酶切位点的内引物。以内外引物进行PCR扩增,将所得PCR产物回收,以相同的限制性内切酶酶切目的基因和表达载体pET-32a后构建重组表达载体,并转入宿主菌BL21和Rosseta,经酶切、PCR和测序鉴定,证明VP1基因正确插入了表达载体。用不同浓度的IPTG诱导VP1基因的表达,收集菌液进行SDS-PAGE电泳,结果表明,VP1基因片段在大肠杆菌Rosseta和BL21中均难以表达。     

建立安全优质高效的饲料生产体系

2005年中央1号文件的主旨是提高农业综合生产能力。增强农业综合生产能力必须培育发达的畜牧业。饲料作为现代畜牧业发展的物质基础,中央1号文件明确要求,加快建立安全优质高效的饲料生产体系。农业部认真贯彻落实中央1号文件精神,制定了相关配套政策和措施,依靠科技,面向市场,     

应用RT—PCR方法检测鸭病毒性肝炎新型和I型病毒

鸭病毒性肝炎病毒是危害养鸭业的重要病毒性传染病,主要感染低日龄雏鸭,临床上分离到的病原主要为鸭病毒性肝炎I型病毒和鸭病毒性肝炎新型病毒,为了加快本病的检测速度,尽快确诊病原,根据GeneBank上两种毒株的基因组序列,选择两种毒株的差异基因序列分别设计了一对特异性的检测引物,将两对引物放在一个PCR反应体系中,两种毒株分别进行特异性扩增,根据扩增片段的大小进行鉴别,建立了可以同时检测鸭病毒性肝炎病毒I型和新型毒株的复合RT—PCR方法,该检测方法快速,特异性强,灵敏性高,可以用于鸭病毒性肝炎病毒的分型鉴别检测。