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.     

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.     

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.     

The hygienic treatment of turkey eggs by dipping in an antibiotic and disinfectant solution

Summary

At the hatchery, in order to reduce bacterial infections in one‐day‐old chicks, turkey eggs are routinely dipped by the direct pressure difference method into a solution consisting of:

tylosin tartrate 2500 ppm

ampicillin sodium 1000 ppm

alkyldimethylbenzylammonium chloride 150 ppm

alkyldimethylethylbenzylammonium chloride 150 ppm

This dipping process was studied in more detail and the experiments allowed of drawing the following conclusions: 1. After 8 hours of use on three consecutive days the tylosin concentiation had hardly decreased, the concentration of ampicillin dropped to approximately 65% and the concentration of quaternary ammonium compounds to 33%.

2. Bacteria were isolated from the fluid only at the time of final sampling (Bacillus subtilis). All other bacteriological investigations of the dipping solution showed negative results.

3. The treatment did not appear to influence hatchhability.

4. The mortality in the first week of life of poults from treated eggs was significantly lower than of poults from untreated eggs.

     

Disinfectants in the control of small animal ringworm due to Microsporum canis

Twelve disinfectant products or compounds were evaluated for their ability to kill Microsporum canis harvested from naturally infected material. The disinfectants were diluted to the concentration recommended for the disinfection of clean surfaces and the potency of each substance was determined by the degree to which it could be further diluted before losing its fungicidal action. Hypochlorite, benzalkonium chloride and glutaraldehyde based compounds were the most effective agents and phenolics, alcohol and anionic detergents were inadequate. Urea (10 mM) did not adversely affect the potency of any of the compounds.     

高寒牧草内生细菌分离培养条件的优化

通过对高寒牧草内生细菌的分离培养方法研究,得出了优化的分离培养条件,即牧草不同组织器官用0．19／6SDS浸泡15min、3％NaClO浸泡3min、0．1％升汞浸泡10min、75％酒精1～2min处理后（各步间均用无菌水冲洗3～4次）,研磨并稀释至10^-3,涂布于TSA培养基上,置28℃恒温条件下培养5—7d,可从高寒牧草组织中分离获得数量、种类较多的内生细菌。用此方法,已从5种牧草不同组织器官（根、茎、叶、花）中分离获得大小、形态、颜色各异的内生细菌315株。     

Safety of Strontium Chloride as a Skeletal Marking Agent for Pacific Salmon

Abstract

The purpose of this study was to evaluate the biological effects associated with administering strontium chloride as a marking agent to age-0 Chinook Salmon Oncorhynchus tshawytscha fry. Fish were held in a 0× (0 mg/L), 1× (3,000 mg/L; current standard dosage), 3× (9,000 mg/L), or 5× (15,000 mg/L) solution of strontium chloride for 72 h (three times the standard duration of 24 h). The mortality among fish in the 5× strontium chloride exposure group was significantly higher than that observed in the other groups. A dose-related effect on general fish behavior and on feeding behavior was observed. Fish in all test tanks appeared to feed to satiation, except for fish in the 5× tanks during days 2 and 3. Fish in all other test tanks behaved normally. No dose-related effect on fish growth was detected. Histopathological evaluations showed that fish in the 5× exposure group had a significantly higher number of gill lesions than the 0× group. Our mortality, behavioral, and histological assessments suggested that juvenile Chinook Salmon could be safely immersed for three consecutive days in a 9,000-mg/L solution of strontium chloride. This finding potentially expands the present 1,000–3,000-mg/L dosage and 24-h holding period that can be used to mark juvenile fish with strontium chloride solutions. The research also provides necessary target animal safety data for U.S. Food and Drug Administration approval of strontium chloride as an alternative marking method that is suitable for fish with a short holding time.

Received February 19, 2017; accepted July 16, 2017 Published online September 22, 2017     

Virulence stability in Flavobacterium psychrophilum after storage and preservation according to different procedures

Experimental infections and lethal dose 50% (LD 50) evaluation were conducted in rainbow trout fingerlings, using a virulent strain of Flavobacterium psychrophilum processed and stored or maintained in different ways; lyophilisation, freezing at -80 degrees C, maintenance in enriched Anacker and Ordal (EAO) medium at 4 degrees C, revival and subsequent in vivo passages in fish. Experiments were performed 1, 8 and 23 months after storing the bacteria. Out of a total of 12 cultures revived for experimentation, one failed to grow and another was found to express modified properties including decreased virulence in spite of in vivo passages. In all other cases, whatever the conditions of preservation, virulence was fairly well maintained after 1 and 8 months of storage. In the last test, after 23 months, the bacteria maintained in the EAO medium at 4 degrees C were found significantly attenuated. Conversely, lyophilised and frozen bacteria only expressed a slight increase in LD0. It was concluded that virulent strains of F. psychrophilum were likely to retain their properties without special provisions within limited periods of time, and that both lyophilisation and freezing at -80 degrees C were reliable methods for long-term preservation of virulence.     

A Comparison of Antimicrobial Efficacy and Tissue Reaction of Four Antiseptics on Canine Wounds

Three concentrations of povidone iodine, chlorhexidine, benzalkonium chloride, and a pluronic polyol were tested for antimicrobial efficacy and tissue reaction in wounds inoculated with Staphyloccocus aureus .
Five paired incisions were created on the back of each of 16 dogs in four treatment groups. Five wounds were inoculated with beta-hemolytic, coagulase positive S. aureus (10⁹ organisms); the contralateral wounds were not inoculated. After two hours, three wounds on each side were irrigated with antiseptic in low, medium, or high concentrations; another was irrigated with physiological saline; and one was left untreated. One hundred ml of solution was applied in each instance with a syringe and needle at approximately 8 psi.
Tissue bacteria were quantitated immediately following irrigation. The wounds were sutured with 5/0 monofilament stainless steel and bandaged. After 48 hours, the dogs were euthanized and signs of wound infection were recorded. Wound bacteria were quantitated again, and sections of the wounds were removed for examination by light microscopy.
Antiseptics were significantly effective (p < 0.01) in reducing the bacterial population in contaminated wounds when compared to control wounds on the same dog. High antiseptic concentrations were more effective than low concentrations. At 48 hours, the bacterial population was significantly lower in wounds treated with chlorhexidine or benzalkonium chloride compared with povidone iodine or pluronic polyol. The antiseptics caused no significant difference (p < 0.01) in tissue reaction in noninoculated wounds. Chlorhexidine (0.5 or 1.0%) was the most effective antiseptic under the conditions used.     

Safe and efficacious dosage of flecainide acetate for treating equine atrial fibrillation

To determine a safe and efficacious dose of flecainide acetate for treating equine atrial fibrillation (Af), the safe dosage level was determined by injecting 1, 2, or 3 mg/kg i.v. of 1% flecainide acetate solution at a rate of 0.2 mg/kg/min to five clinically healthy horses. Clinical signs and the ECG were monitored (HR, PR, QRS, and QT intervals) and blood was taken to measure the plasma flecainide concentration pre- and post-administration. No abnormal signs were observed in the 1- or 2-mg/kg groups, while agitation was observed in three of five horses in the 3-mg/kg group. The QRS, and QT intervals for the 3-mg/kg group increased significantly. The peak plasma flecainide concentrations were 1.316 +/- 358 (SD) ng/ml, 1,904 +/- 314 ng/ml, and 2,251 +/- 387 ng/ml for the 1-, 2-, and 3-mg/kg groups, respectively. To evaluate the efficacy of flecainide, Af was induced by right atrial pacing in six clinically healthy horses, and 1% flecainide acetate solution was then administered until they converted to sinus rhythm. All horses with induced Af converted. For the conversion, a total dose of 1.40 +/- 0.63 mg/kg flecainide was required, the duration of administration was 7.00 +/- 3.15 min and plasma flecainide concentration at conversion was 1,303 +/- 566 ng/ml. In conclusion, flecainide acetate is a safe and effective antiarrhythmic agent for equine Af, and the clinically effective dosage is 1 to 2 mg/kg.     

野牛草成熟胚植株再生及其影响因素研究

以野牛草Buchloe dactyloides成熟胚为外植体,对外植体灭菌方式及影响成熟胚再生的因素进行了研究.结果表明:70%酒精处理60 s,75% NaClO溶液(原溶液含有效氯为7%～10%)处理30 min,污染率最低为0.59%.6-BA 0.1 mg/L,2,4-D浓度增至3 mg/L时,愈伤组织诱导率达最高,为80.27%,但愈伤组织结构疏松、呈水渍状.附加5 mg/L硝酸银(AgNO3)或硫代硫酸银(STS)时,愈伤组织诱导率略有降低,愈伤组织结构紧密且表面有颗粒状突起,当AgNO3或 STS浓度为10 mg/L以上时,均不利于愈伤组织的诱导.愈伤组织继代培养过程中,3/4倍MS大量元素用量、聚乙烯吡咯烷酮(PVP) 200 mg/L、维生素C(Vc )200 mg/L及水解酪蛋白(CH) 1 000 mg/L可减轻愈伤组织褐化程度.不同诱导培养基所获得的愈伤组织与其植株再生能力有关,以不加任何植物生长调节剂的MS培养基(MS0)为分化培养基,仅在附加STS的诱导培养基中所得的愈伤组织能够形成再生植株,再生率为10%.     

Disinfectant susceptibility testing of avian and swine Campylobacter isolates by a filtration method

The susceptibility testing of disinfectants against Campylobacter jejuni and Campylobacter coli strains from broilers and pigs was investigated. The filtration method European standard EN 1040 was adapted to Campylobacter cultures and validated with reference strains. Two disinfectants were tested: 1% benzalkonium chloride active matter, as quaternary ammonium compound, and 0.63% sodium hypochlorite as chlorine-releasing agent. Both disinfectants were effective against the 34 Campylobacter strains tested after 5 min exposure under in vitro conditions. No link between resistance to disinfectants and antibiotics could be observed.     

Toxicity of Copper Sulfate to Flavobacterium psychrophilum and Rainbow Trout Eggs

Abstract

Tests were conducted to determine the concentrations of copper sulfate needed to kill Flavobacterium psychrophilum, the cause of bacterial coldwater disease, either in vitro or on Rainbow Trout Oncorhynchus mykiss eggs. For the in vitro test, a plastic strip dipped in a solution of F. psychrophilum was exposed for 15 min to copper sulfate solutions of 0, 1, 5, 10, 20, 35, 50, 75, or 100 mg/L. Bacteria were “too numerous to count” at concentrations ≤10 mg/L CuSO₄; significant reductions in prevalence relative to untreated controls were noted for concentrations ≥35 mg/L. However, CFUs were still observed at 50 and 75 mg/L (20% of plates with tryptone yeast extract salts media). No yellow-pigmented CFUs typical of F. psychrophilum were observed at 100 mg/L CuSO₄. For the in vivo test, eggs were exposed for 15 min to 100, 300, 500, and 700 mg/L CuSO₄ or 100 mg/L iodine (control). Survival to hatch was significantly lower at 500 (44.3 ± 15.2%, mean ± SD) or 700 mg/L CuSO₄ (1.7 ± 0.8%) than for controls treated with 100 mg/L iodine (93.6 ± 0.9%) or at copper sulfate concentrations ≤300 mg/L. The 15-min LD₅₀ and LD₁₀ for copper sulfate were 461 mg/L (95% confidence interval: 457–466 mg/L) and 259 mg/L (251–266 mg/L). The prevalence of yellow CFUs at 100 mg/L CuSO₄ (40.0%) was significantly higher than in untreated controls. Significant reductions in yellow CFUs were achieved using 300, 500, or 700 mg/L CuSO₄ (7.5, 2.5, or 0.0% of plates with CFUs, respectively) or 100 mg/L iodine (2.5%), relative to untreated control eggs. Overall, since the concentrations of copper sulfate required to eliminate F. psychrophilum were toxic to the eggs, copper sulfate is not recommended for coldwater disease control in Rainbow Trout eggs based on conditions and parameters in this study.

Received July 7, 2011; accepted March 17, 2013     

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.     

Disinfection of the causative agent of contagious equine metritis by Nolvasan and Roccal II

Haemophilus equigenitalis, the proposed name for the bacterium that causes contaginous equine metritis (CEM) was tested for sensitivity to the disinfectant solutions Nolvasan® (2% chlorhexidine diacetate) and Roccal II® (10 alkyldimethylbenzylammonium chloride).Bacteria (10⁶) suspended in medium were inactivated in 10–20 min at 0°–2°C and in 2.5 min at 20°–22°C by a 3 : 128 dilution of Nolvasan. Roccal II, diluted 1 : 128, inactivated 10⁶ bacteria in 2.5 min at 0°–2°C and 20°–22°C.These dilutions of the disinfectants inactivated H. equigenitalis (titers of 0.1–11.0 × 10⁷/ml) in vaginal exudates from infected mares and pure cultures suspended in medium (titers of 1–5 × 10⁹/ml) in 10 min at 0°–2°C and 20°–22°C when the exudates and cultures were dried on metal carriers. The limit of detection of survivors was 3–32 bacteria/ml.It is recommended that contaminated instruments and other metal surfaces encountered during CEM infections should be decontaminated with either disinfectant for 10 min before rinsing.     

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.     

Cultural and biochemical characteristics of Mycobacterium paratuberculosis isolated from goats in Norway.

In Norway a variant of Mycobacterium paratuberculosis occurs which causes disease in goats but very seldom in sheep and cattle. Cultural and biochemical characteristics of this variant are investigated by comparing different pre-treatment methods and culture media for primary isolation and by subjecting a number of strains to different enzymatic and biochemical tests. Decontamination of materials with 5% oxalic acid and 0.1% benzalkonium chloride and culture on Dubos, Finleyson’s and Herrold’s medium was tested. The investigations showed that the combination oxalic acid decontamination/Dubos’ medium is most suitable for isolation of the goat-pathogenic variant.The morphology of the colonies was also most easily studied after culture on Dubos’ medium from material pre-treated with oxalic acid. The biochemical tests were found to be poorly suitable for the identification of M. paratuberculosis and for its differentiation from other mycobacteria.Mycobactin dependence for growth seems not to be absolute as a few goat strains produced growth on Dubos’ medium without mycobactin. However, growth was in all cases far better in the presence of mycobactin.     

高羊茅胚性愈伤组织的高效诱导及其耐盐突变体筛选

以高羊茅Fine-lawn种子和下胚轴为外植体,分别在诱导培养基D1、D3、D5和D9上培养.下胚轴愈伤组织的出愈率除了在D1培养基上为51.3%外,在其他诱导培养基上均为100%.在D5培养基上继代3个月左右,从下胚轴获得了胚性愈伤组织.取在NaCl浓度为0.5%～3.0%的培养基上生长13 d的高羊茅胚性愈伤组织,分别测定其存活率、相对生长量、鲜干重比和脯氨酸含量.利用直接筛选的方法,将胚性愈伤组织分别放在含1%, 2%和3% NaCl的选择培养基上连续筛选60 d,在1% NaCl浓度下获得了耐盐的胚性愈伤组织并且获得了再生植株.再生植株能够在含有1% NaCl的Hoagland培养液中生长.     

Rhodococcus equi: equine neutrophil chemiluminescent and bactericidal responses to opsonizing antibody

The opsonic capacity of serum containing R. equi-specific antibody was compared with antibody-deficient sera using luminol-dependent chemilumenscence (LDCL) and bactericidal assays. These assays incorporated peripheral blood polymorphonuclear neutrophilic leukocytes (PMNL) exposed to R. equi opsonized with neonatal equine pre-colostral serum (control) or serum from foals with R. equi infections (principal). All sera were complement inactivated at 56 degrees C for 30 min. Bacteria were obtained from the lung of a foal with R. equi pneumonia. Neutrophils were obtained from one adult horse for LDCL and another for bactericidal assays. Chemiluminescence of PMNL exposed to R. equi opsonized with control or principal sera was measured in a liquid scintillation counter. Mean peak LDCL within 1 h was significantly (P less than 0.01) higher with principal sera (2.4 X 10(5) cpm) than with control sera (0.018 X 10(5) cpm). A radioisotope bactericidal assay was used to determine the effect of control or principal sera on PMNL capacity to kill R. equi. Mean peak percent kill of R. equi by PMNL within 2 h was significantly (P less than 0.01) higher with principal sera (95.2%) than with control sera (54.6%). Enzyme-linked immunosorbent assay (ELISA) values for R. equi-specific antibody were determined on all sera. Mean ELISA values were significantly (P less than 0.01) higher for principal sera (71.8) than for controls (0.0). This investigation documents the presence and biological effectiveness of opsonic activity in complement-inactivated sera from foals with R. equi infections and R. equi-specific antibody.