In vitro competitive adhesion and production of antagonistic compounds by lactic acid bacteria against fish pathogens

The present study describes the screening of five lactic acid bacteria (LAB) for use as probiotics based on their competitive adhesion and production of antagonistic substances against some fish pathogens. A reduction of adhesion of all pathogenic strains tested was obtained with three of the LAB strains (Lactococcus lactis subsp. lactis CLFP100, Lactococcus lactis subsp. cremoris CLFP102 and Lactobacillus curvatus CLFP150). With the exception of fish pathogens Flavobacterium psychrophilum and Renibacterium salmoninarum that were not inhibited by LAB strains, production of antagonistic compounds by all tested LAB was observed against at least one of the indicator strains. Based on mucus adhesion, competitive exclusion, and suppression of fish pathogen growth, the selected LAB strains can be considered for future challenge experiments in fish as a very promising alternative to the use of chemotherapeutic agents.     

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.     

Concomitant exposure of rainbow trout fry to Gyrodactylus derjavini and Flavobacterium psychrophilum: effects on infection and mortality of host

Although rainbow trout fry mortality syndrome caused by the bacterium Flavobacterium psychrophilum is widespread in fish farms it is difficult to reproduce infection of rainbow trout in the laboratory using immersion exposure with bacterial suspensions. It has therefore been speculated that ectoparasites could act as enhancers of bacterial infections under natural conditions. In the present study rainbow trout fry were exposed to infections with F. psychrophilum (immersion for 30 min or 10 h) alone, exposed to the ectoparasitic monogenean Gyrodactylus derjavini alone or exposed to both pathogens in combination. Infection levels and host mortality were subsequently monitored to elucidate if the ectoparasitic monogeneans could enhance infection of fish with the bacterium. Immersion of fish in bacterial suspensions alone did not result in infection. Only one fish became infected with the bacterium and this fish belonged to the combination exposure group. The parasite populations increased differently in the various groups and it was found that host mortality was correlated to gyrodactylid infection levels (r=0.94) but not to bacterial exposure. The results emphasise the pathogenicity of the parasite G. derjavini, the relative resistance of intact fish to direct exposure to F. psychrophilum but provide only a weak indication of a possible enhancement of bacterial invasion due to ectoparasitic infections. It cannot be excluded that higher parasite burdens and/or prolonged immersion (more than 10 h) in bacterial suspensions may result in bacterial invasion.     

Improved pulsed-field gel electrophoresis procedure for the analysis of Flavobacterium columnare isolates previously affected by DNA degradation

Flavobacterium columnare is a fresh water bacterium that causes columnaris diseases in over 36 fish species. Intra-species typing of F. columnare can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 10% of strains. In the current study, DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. The results substantiate that after problems due to DNases are overcome, PFGE analysis is a reproducible highly discriminating epidemiological method for studying F. columnare isolates regardless of fish host.     

Biofilm Formation on Aquaculture Substrates by Selected Bacterial Fish Pathogens

Abstract

The objective of this study was to determine whether common bacterial catfish pathogens could attach and colonize surfaces commonly found in aquaculture facilities. In addition, we evaluated the role of calcium in biofilm formation. Attachment to polystyrene plates was used to quantify biofilm formation by five bacterial pathogens (i.e., Flavobacterium columnare, Aeromonas hydrophila, Edwardsiella ictaluri, E. tarda, and E. piscicida). Flavobacterium columnare and A. hydrophila formed thick biofilms that were enhanced by calcium supplementation. Biofilm formation was significantly lower in all Edwardsiella species tested and calcium had little to no effect on Edwardsiella biofilm formation. Attachment to natural and artificial surfaces was quantified by a standard plate count method. Scanning electron microscopy (SEM) was used to confirm biofilm formation on the substrates. Flavobacterium columnare formed biofilm on the liner, flexible PVC, and nets. Bamboo prevented F. columnare attachment and inhibited cell growth. Aeromonas hydrophila and E. ictaluri formed biofilm on all materials tested, although significant differences were found among substrates. While E. ictaluri failed to form biofilm on microtiter polystyrene plates, it was able to colonize and multiply on all aquaculture materials tested. Our results demonstrated that common bacterial pathogens had the potential of colonizing surfaces and may use biofilm as reservoirs in fish farms.

Received July 19, 2016; accepted January 19, 2017 Published online April 13, 2017     

Characterization of the probiotic strain Vagococcus fluvialis in the protection of European sea bass (Dicentrarchus labrax) against vibriosis by Vibrio anguillarum

Aquaculture is one of the main sources of income in many countries worldwide. Intensive farms are often affected by different infectious diseases that can decrease their final production. To control this situation, several antibiotics are frequently used with known environmental consequences. The aim of this study was to analyze different bacterial strains isolated from of gilthead sea bream, sea bass, sole and meagre guts, for use as probiotics in aquaculture. The strains were evaluated in vitro through various mechanisms of selection, such as the production of antagonistic effects against pathogens, production of antibacterial substance, adhesion to the intestinal mucus, competition for nutrients or binding site, and growth in intestinal mucus. A total of 50 bacterial strains were analyzed and only one showed excellent in vitro results for consideration as a candidate to be analyzed in vivo. The strain, identified as Vagococcus fluvialis, showed good protection against Vibrio anguillarum 975-1 in vivo in the experimental challenge, showing a relative percent survival of 42.3% higher than positive control group. Therefore, in conclusion we consider this strain to be a good candidate for use as a future probiotic in aquaculture.     

Potassium permanganate elicits a shift of the external fish microbiome and increases host susceptibility to columnaris disease

The external microbiome of fish is thought to benefit the host by hindering the invasion of opportunistic pathogens and/or stimulating the immune system. Disruption of those microbial communities could increase susceptibility to diseases. Traditional aquaculture practices include the use of potent surface-acting disinfectants such as potassium permanganate (PP, KMnO₄) to treat external infections. This study evaluated the effect of PP on the external microbiome of channel catfish and investigated if dysbiosis leads to an increase in disease susceptibility. Columnaris disease, caused by Flavobacterium columnare, was used as disease model. Four treatments were compared in the study: (I) negative control (not treated with PP nor challenged with F. columnare), (II) treated but not challenged, (III) not treated but challenged, and (IV) treated and challenged. Ribosomal intergenic spacer analysis (RISA) and pyrosequencing were used to analyze changes in the external microbiome during the experiment. Exposure to PP significantly disturbed the external microbiomes and increased catfish mortality following the experimental challenge. Analysis of similarities of RISA profiles showed statistically significant changes in the skin and gill microbiomes based on treatment and sampling time. Characterization of the microbiomes using 16S rRNA gene pyrosequencing confirmed the disruption of the skin microbiome by PP at different phylogenetic levels. Loss of diversity occurred during the study, even in the control group, but was more noticeable in fish subjected to PP than in those challenged with F. columnare. Fish treated with PP and challenged with the pathogen exhibited the least diverse microbiome at the end of the study.     

The effect of high total ammonia concentration on the survival of channel catfish experimentally infected with Flavobacterium columnare

Ammonia concentrations in water can affect the severity of Flavobacterium columnare infections in fish. Two trials lasting 7 d each were conducted to determine the effect of a single immersion flush treatment of total ammonia nitrogen (TAN; 15 mg/L) on the survival of channel catfish Ictalurus punctatus infected with E columnare; the chemical was added while the water flowed continuously through the tanks. Both trials consisted of four treatments: (1) no ammonia exposure and no bacterial challenge (control), (2) ammonia exposure only, (3) bacterial challenge only, and (4) both ammonia exposure and bacterial challenge. Two hours after exposure to ammonia, the highest un-ionized ammonia level was 0.43 mg/L. The percent un-ionized ammonia is based on TAN, temperature, and pH. Caudal fins from three fish in each treatment were sampled at 24 h posttreatment to be analyzed by quantitative real-time polymerase chain reaction (qPCR). No significant difference in survival (mean +/- SE) was noted between the channel catfish in treatment 1 (95.2 +/- 1.2%) and those in treatment 2 (95.6 +/- 1.0%); however, survival in both treatments 1 and 2 differed significantly from that in treatments 3 (8.5 + 4.5%) and 4 (41.8 +/- 12.7%). Treatment 4 catfish had significantly higher survival than treatment 3 catfish. Quantitative PCR data showed that treatment 4 fish had significantly less F. columnare (7.6 x 10(5)) than did treatment 3 fish (1.2 x 10(7)), and treatment 2 fish (8.5 x 10(3)) had significantly less bacteria than did treatment 1 fish (6.9 x 10(4)), indicating that ammonia limited the F. columnare infection. The highest mean concentration of the bacteria (3.9 x 10(7)) was found on moribund fish. The ammonia concentrations tested did not negatively influence fish survival but interfered with the infection process. An in vitro assay was also conducted to evaluate the direct effects of ammonia on F columnare.     

Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens

A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method.     

In vitro study of adherence, invasion, and persistence of Streptococcus iniae in fibroblastic-like fish cell line SAF-1

Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Gentamicin protection assays were used to investigate the ability of different S. iniae strains to invade and adhere to fibroblastic-like fish cell line SAF-1. All strains tested were detected intracellularly using both techniques, with variable internalization degrees between strains. The experiments carried out at 4°C demonstrated that active cell metabolism is necessary for bacterial internalization. Intracellular bacteria were detected for up to 3 d with a round morphology and were stained with 4',6-diamidino-2-phenylindole (DAPI), indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that S. iniae is capable of adhering, entering, and surviving within fibroblastic cells, which may be important for the persistence and establishment of a carrier state.     

Journal of Aquatic Animal Health: Guide for Authors 2014

Abstract

Brook trout Salvelinus fontinalis were treated with single 60-min static baths of 250 mg formalin/L, 3% NaCl, and 15 mg Chloramine-T/L to evaluate the efficacy of these compounds against external infections of Aeromonas salmonicida. Prevalence of A. salmonicida was significantly lower in brook trout treated with Chloramine-T than among those treated with formalin or salt. Further laboratory tests substantiated the therapeutic value of a single treatment of ChloramineT (15 mg/L) against A. salmonicida. In two experiments, viable counts of A. salmonicida in mucus did not vary among replicate groups of treated brook trout, but the counts for treated fish were significantly (P < 0.05) lower than those for untreated controls. In vitro tube dilution assays indicated that mean minimum inhibitory concentrations of Chloramine-T for 10 isolates of A. salmonicida were 9.0 mg/L for 1 h and 2.25 mg/L, for 24 h. In field trials at the White River National Fish Hatchery (Bethel, Vermont), the pathogen was detected principally as an external infection of juvenile Atlantic salmon Salmo solar maintained in two culture ponds. In one pond, the bacterium accounted for 100% of the total distribution of tnicroflora isolated from mucus. Seven days after treatment with Chloramine-T, A. sahnonicida accounted for 11% of the total bacterial counts identified from these fish. In the second pond, A. salmonicida composed 3% of the counts of bacteria isolated from the mucus of fish before treatment but was not isolated after treatment.     

水生动物柱状黄杆菌的研究进展

柱状黄杆菌是柱形病的病原体,可感染全球的淡水鱼类。自然和养殖条件下的海水鱼类以及观赏鱼类也可感染发病。全世界每年由柱形病所造成的经济损失极其严重。文章回顾和总结了柱状黄杆菌的发现、病理变化、超微结构特点和血液学特征等方面的研究近况,对柱状黄杆菌的分离鉴定、柱形病的爆发条件及防治措施进行了概述,并提出了柱状黄杆菌研究的发展方向。     

枯草芽孢杆菌MA139类细菌素抑菌活性的研究

为测定枯草芽孢杆菌MA139产生的类细菌素的抑菌活性。本试验采用管碟法对枯草芽孢杆菌MA139产生的类细菌素对多株革兰氏阳性菌、革兰氏阴性菌和真菌的抑菌活性进行测定。结果表明:类细菌素不但对芽孢杆菌属的其他菌种有抗菌活性,对病原菌和霉菌也有拮抗作用;不同指示菌对类细菌素的敏感性不一样,其中金黄色葡萄球菌对其最敏感。该类细菌素具有广谱抗菌作用,有作为畜禽饲料添加剂的潜力。     

Development and evaluation of an experimental model of cutaneous columnaris disease in koi Cyprinus carpio.

A reproducible, experimental model of columnaris disease was developed to study the pathogenesis of cutaneous disease associated with Flavobacterium columnare infection in koi (Cyprinus carpio). In experimental infections, lesions were usually restricted to skin and fins; gill necrosis was not a consistent finding. Cytologic and histopathologic examinations provided a presumptive diagnosis of columnaris disease. Specific detection of F. columnare was done using the polymerase chain reaction and DNA in situ hybridization (ISH). Polymerase chain reaction allowed the detection of F. columnare in fresh biological material and in formalin-fixed, paraffin-embedded tissues. The DNA ISH technique allowed the identification and localization of F. columnare in formalin-fixed, paraffin-embedded tissues. Using these molecular techniques, F. columnare was readily detected in skin specimens from infected fish; however, the bacterium was infrequently detected in specimens of liver, kidney, and spleen. These observations suggest that columnaris disease generally presents as a cutaneous disease that is unassociated with systemic infection in koi. Hematologic studies indicated that most infected koi developed microcytic, normochromic, nonregenerative anemia and leukopenia characterized by lymphopenia, mild neutrophilia, and monocytosis. Biochemical changes in diseased fish included significant hyperglycemia, hyponatremia, and hypochloridemia.     

Difficulties in experimental infection studies with Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) using immersion, oral and anal challenges

This study was carried out in order to try to establish an efficacious and reliable experimental infection model for Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome, using contact, oral and anal challenges. Ten F psychrophilum strains of different origin were included. The influence of water temperature, scarification, water quality, stress and growth conditions of the pathogen on the experimental infection was assessed. For each challenge protocol, all strains failed to reproduce disease signs or mortality in rainbow trout (Oncorhynchus mykiss L.) fry. Histological and bacteriological examination of the skin, gills and internal organs of the fish 3 weeks following inoculation were found to be negative. Different hypotheses to explain the inability of the challenge models to reproduce the disease experimentally are discussed.     

In Vitro Study of Adherence,Invasion, and Persistence of Streptococcus iniae in Fibroblastic-Like Fish Cell Line SAF-1

Abstract

Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Gentamicin protection assays were used to investigate the ability of different S. iniae strains to invade and adhere to fibroblastic-like fish cell line SAF-1. All strains tested were detected intracellularly using both techniques, with variable internalization degrees between strains. The experiments carried out at 4°C demonstrated that active cell metabolism is necessary for bacterial internalization. Intracellular bacteria were detected for up to 3 d with a round morphology and were stained with 4′,6-diamidino-2-phenylindole (DAPI), indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that S. iniae is capable of adhering, entering, and surviving within fibroblastic cells, which may be important for the persistence and establishment of a carrier state.

Received July 8, 2011; accepted February 12, 2012     

Use of Eggs Derived From the Interspecific Charr Hybrids to Induce Androgenetic Development of the Brook Charr (Salvelinus fontinalis Mitchill 1814)

Although, brook charr (Salvelinus fontinalis Mitchill 1814) and Arctic charr (Salvelinus alpinus Linnaeus 1758) are able to cross and give fertile offspring, their androgenetic nucleocytoplasmic hybrids are not viable. To overcome incompatibility between the egg cytoplasm of one charr species and the sperm nucleus of another charr species, application of F₁ interspecific hybrids as egg donors for the purpose of androgenesis has been proposed. Here, androgenetic development of the brook charr was successfully induced in the brook charr eggs and the eggs derived from the reciprocal brook charr × Arctic charr F₁ hybrids. A working androgenesis protocol included inactivation of the maternal nuclear DNA achieved by irradiation of the eggs with 420 Gy of X‐rays, insemination of such treated eggs with the haploid sperm cells and exposition of the haploid androgenetic zygotes to the high hydrostatic pressure shock (51.711 MPa for 4 min) applied 420 min after insemination. Androgenetic larvae that hatched from the brook charr and the hybrid eggs were shown to be homozygous brook charr individuals. Androgenetic individuals exhibited 84 chromosomes and 100 chromosome arms (FN), values characteristic for the brook charr diploid cells. Strategy hybridize first than induce androgenesis should be tested in order to provide androgenetic offspring in other salmonids that are able to cross and produce fertile offspring.     

Growth Inhibition of Bacterial Fish Pathogens and Quorum-Sensing Blocking by Bacteria Recovered from Chilean Salmonid Farms

The main goal of this study was to find bacterial isolates with the ability to inhibit the growth of the fish pathogens Aeromonas hydrophila, Vibrio anguillarum, and Flavobacterium psychrophilum and to inhibit the blockage of the quorum-sensing (QS) system. A total of 80 gram-negative strains isolated from various freshwater Chilean salmonid farms were studied. We determined that 10 strains belonging to the genus Pseudomonas inhibited at least one of the assayed fish pathogens. Of these, nine strains were able to produce siderophores and two strains were able to inhibit the growth of all assayed pathogenic species. When the 80 strains were examined for QS-blocking activity, only the strains Pseudomonas sp. FF16 and Raoultella planticola R5B1 were identified as QS blockers. When the QS-blocker strains were analyzed for their ability to produce homoserine lactone (HSL) molecules, thin-layer chromatography analysis showed that both strains were able to produce C6-HSL– and C8-HSL–type molecules. Strain R5B1 did not show growth inhibition properties, but strain FF16 also led to inhibition of growth in A. hydrophila and F. psychrophilum as well as to siderophore production. Pseudomonas sp. FF16 exhibited potentially useful antagonistic properties and could be a probiotic candidate for the salmon farming industry.

Received July 31, 2014; accepted December 17, 2014     

Effects of media ingredient substitution and comparison of growth of Flavobacterium psychrophilum among four media

The etiological agent of bacterial cold-water disease, Flavobacterium psychrophilum, can cause significant losses of salmonid fishes in aquaculture facilities. Few studies describing the value of media components on the growth of F. psychrophilum are available in the literature. We therefore conducted a study that began with the standard enriched Anacker-Ordal broth (EAO) and over the course of multiple iterations evaluated the effects of various media supplements by adding or subtracting them from the base EAO medium. Different media formulations were made, and samples were removed from each broth formulation every 24 h for 72 h. From those samples we determined bacterial density by measuring absorbance values with a spectrophotometer. The medium with the highest absorbance value from one iteration was used as the base medium in the next iteration. Using this iterative approach, we determined that sodium acetate, calcium chloride, and magnesium sulfate inhibit growth and that maltose has no effect on the proliferation of the bacterium. The addition of skimmed milk (0.2%) and horse serum (1%) appears to provide a slight improvement in bacterial proliferation. Variations in agar concentration had no effect on the growth of the bacterium. Even though the addition and removal of some ingredients increased the mean absorbance values, the benefit of these substitutions was not significant. Even so, we found that the growth of F. psychrophilum in EAO was better than that in two other widely used media: tryptone-yeast extract salts and maltose infused tryptone-yeast extract salts.     

木霉菌对草坪草病原菌的抑菌效果及其机理初步研究

木霉菌是自然界中普遍存在的拮抗微生物,多种木霉菌对植物病原菌具有重要的生防价值。为挖掘出防治草坪草病害的生防菌剂,采用对峙培养法测定了4株木霉菌株对14种草坪草病原菌的拮抗作用,通过显微镜观察、玻璃纸法及粗提液抑菌试验研究了其抑菌机理。研究结果表明,供试木霉菌对所有供试病原菌均有拮抗作用,其中菌株SQ-1Q-18抑菌效果最佳,对玉蜀黍丝核菌和地衣状伏革菌的拮抗等级为I级,抑制率高达100%,对灰葡萄孢菌、地毯草炭疽菌和立枯丝核菌的拮抗等级为Ⅱ级,抑制率大于80%,且对供试病原菌的平均抑制率高达72.8%。进一步研究菌株SQ-1Q-18的抑菌机理发现,该菌株在培养基上产生抑菌圈侵占病原菌的生长空间,覆盖和深入病原菌内部并与其相互缠绕使得病原菌菌丝变细、缢缩甚至断裂,或直接穿入病原菌菌丝内部吸取营养致使菌丝细胞溶解,也可产生拮抗物质,其中玻璃纸法中对草坪草币斑病菌、球黑孢霉及地衣状伏革菌的相对抑制率分别高达91.77%、89.80%和79.59%;粗提取液对草坪草币斑病菌和佩立金平脐蠕孢的抑制率分别为43.60%和42.91%。经鉴定,SQ-1Q-18为哈茨木霉。研究结果为高效防治草坪草真菌病害的生防菌剂的研制、开发提供了科学资料。