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     

Predictive Factors of Sensitivity to Elisidepsin,a Novel Kahalalide F-Derived Marine Compound

Elisidepsin (PM02734, Irvalec^®) is a synthetic marine-derived cyclic peptide of the Kahalalide F family currently in phase II clinical development. Elisidepsin was shown to induce rapid oncosis in ErbB3-expressing cells. Other predictive factors of elisidepsin sensitivity remained unknown. A panel of 23 cancer cell lines of different origin was assessed for elisidepsin cytotoxicity and correlated with mutational state, mRNA and protein expression of selected genes. Elisidepsin showed potent and broad cytotoxic effects in our cancer cell line panel, being active at concentrations ranging from 0.4 to 2 μM that may be relevant for clinical settings. We have shown that elisidepsin is more active in cells harboring epithelial phenotype with high E-cadherin and low vimentin expression. In addition, high ErbB3 and Muc1 expression was correlated with sensitivity to elisidepsin, whereas the presence of KRAS activating mutations was associated with resistance. In DU-PM cells with acquired resistance to elisidepsin, ErbB3 expression was decreased, while Bcl2 was increased. DU-PM cells displayed higher sensitivity to ErbB1-inhibitors suggesting possible cross-talk of ErbB1 and ErbB3 signaling pathways. Combinations of elisidepsin with lapatinib and several chemotherapies including 5-FU and oxaliplatin resulted in synergistic effects that offer the potential of clinical use of elisidepsin in combination settings.     

Population structure of the fish pathogen Flavobacterium psychrophilum at whole-country and model river levels in Japan

The bacterium Flavobacterium psychrophilum is a serious problem for salmonid farming worldwide. This study investigates by multilocus sequence typing (MLST) the population structure of this pathogen in Japan where it is also a major concern for ayu, a popular game fish related to salmoniforms. A total of 34 isolates collected across the country and 80 isolates sampled in a single model river by electrofishing were genotyped. The data accounting for 15 fish species allowed identifying 35 distinct sequence types (ST) in Japan. These ST are distinct from those reported elsewhere, except for some ST found in rainbow trout and coho salmon, two fish that have been the subject of intensive international trade. The pattern of polymorphism is, however, strikingly similar across geographical scales (model river, Japan, world) in terms of the fraction of molecular variance linked to the fish host (~50%) and of pairwise nucleotide diversity between ST (~5 Kbp^-1). These observations go against the hypothesis of a recent introduction of F. psychrophilum in Japan. Two findings were made that are important for disease control: 1) at least two independent F. psychrophilum lineages infect ayu and 2) co-infections of the same individual fish by different strains occur.     

Dynamics of influenza A virus infections in permanently infected pig farms: evidence of recurrent infections,circulation of several swine influenza viruses and reassortment events

Concomitant infections by different influenza A virus subtypes within pig farms increase the risk of new reassortant virus emergence. The aims of this study were to characterize the epidemiology of recurrent swine influenza virus infections and identify their main determinants. A follow-up study was carried out in 3 selected farms known to be affected by repeated influenza infections. Three batches of pigs were followed within each farm from birth to slaughter through a representative sample of 40 piglets per batch. Piglets were monitored individually on a monthly basis for serology and clinical parameters. When a flu outbreak occurred, daily virological and clinical investigations were carried out for two weeks. Influenza outbreaks, confirmed by influenza A virus detection, were reported at least once in each batch. These outbreaks occurred at a constant age within farms and were correlated with an increased frequency of sneezing and coughing fits. H1N1 and H1N2 viruses from European enzootic subtypes and reassortants between viruses from these lineages were consecutively and sometimes simultaneously identified depending on the batch, suggesting virus co-circulations at the farm, batch and sometimes individual levels. The estimated reproduction ratio R of influenza outbreaks ranged between 2.5 [1.9-2.9] and 6.9 [4.1-10.5] according to the age at infection-time and serological status of infected piglets. Duration of shedding was influenced by the age at infection time, the serological status of the dam and mingling practices. An impaired humoral response was identified in piglets infected at a time when they still presented maternally-derived antibodies.     

Towards a better understanding of Rift Valley fever epidemiology in the south-west of the Indian Ocean

Rift Valley fever virus (Phlebovirus, Bunyaviridae) is an arbovirus causing intermittent epizootics and sporadic epidemics primarily in East Africa. Infection causes severe and often fatal illness in young sheep, goats and cattle. Domestic animals and humans can be contaminated by close contact with infectious tissues or through mosquito infectious bites. Rift Valley fever virus was historically restricted to sub-Saharan countries. The probability of Rift Valley fever emerging in virgin areas is likely to be increasing. Its geographical range has extended over the past years. As a recent example, autochthonous cases of Rift Valley fever were recorded in 2007–2008 in Mayotte in the Indian Ocean. It has been proposed that a single infected animal that enters a naive country is sufficient to initiate a major outbreak before Rift Valley fever virus would ever be detected. Unless vaccines are available and widely used to limit its expansion, Rift Valley fever will continue to be a critical issue for human and animal health in the region of the Indian Ocean.     

Silage additives to reduce protein degradation during ensiling and evaluation of in vitro ruminal nitrogen degradability

Despite the high degradability of their proteins, grass and legume silages represent an important option to reach more sustainable livestock systems. To improve the nitrogen use efficiency of these crops, this study assessed the potential of several additives (chestnut tannins, oak tannins, zeolite, erythritol by‐product solution and wood molasses) to reduce proteolysis in the silo and in vitro nitrogen degradability. Ryegrass (Lolium multiflorum) and red clover (Trifolium pratense) were ensiled in varying proportions in laboratory‐scale silos made of vacuum‐packed plastic bags. Dry‐matter content, chemical composition, pH, ammonia and volatile fatty acids content were analysed after 34 days of ensiling. Ruminal nitrogen degradability was assessed in vitro (Aufrère & Cartailler, 1988 ). We observed that the proportion of ammonia in silage was reduced by the addition of oak tannin (?12%) and zeolite (?16%). The addition of zeolite lowered in vitro organic matter digestibility. Rapidly degradable nitrogen (1‐hr degradability) was reduced in vitro by both tannins (?6.8% for chestnut and ?6.6% for oak) and zeolite (?5.8%), but total degradable nitrogen (24‐hr degradability) was only reduced by oak (?6.5%) and chestnut tannins (?7.3%). It suggests that tannins protected proteins from plant and bacterial enzymes by forming a complex that better resists silage fermentations and in vitro protease action. The reduction effects on proteolysis in the silo and on in vitro ruminal nitrogen degradability are limited individually but could be cumulative. Erythritol by‐product solution and wood molasses had no effect on silo or in vitro proteolysis.     

Effect of heating oxymyoglobin and metmyoglobin on the oxidation of muscle microsomes

Myoglobin (Mb) and its iron have been proposed to be major prooxidants in cooked meats. To understand the mechanisms and differentiate between the prooxidant and antioxidant potential of oxymyoglobin (OxyMb) and metmyoglobin (MetMb), their prooxidant activity, iron content, solubility, free radical scavenging activity, and iron binding capacity were determined as a function of thermal processing. The ability of native and heat denatured OxyMb and MetMb to promote the oxidation of muscle microsomes was different. MetMb promoted lipid oxidation in both its native and denatured states. Conversely, OxyMb became antioxidative when the protein was heated to temperatures >or=75 degrees C. The increased antioxidant activity of heat denatured OxyMb was likely due to a decrease in its prooxidative activity due to its loss of solubility. These data show that the impact on oxidative reactions of Mb is the result of the balance between its antioxidant and prooxidant activities.     

New insights on 3-mercaptohexanol (3MH) biogenesis in Sauvignon Blanc wines: Cys-3MH and (E)-hexen-2-al are not the major precursors

The molar conversion yield of Cys-3MH into 3MH, during alcoholic fermentation, was traced using a deuterated isotope of the precursor added to different Sauvignon Blanc musts. This yield is close to that found in synthetic media supplemented with synthetic Cys-3MH, that is, below 1%. Yet, this represents only 3-7% of the total 3MH production in wine. This clearly shows that Cys-3MH is a precursor of 3MH, but not the main one in the different musts tested. The contribution of ( E)-hex-2-enal, which has been suggested as another potential precursor of 3MH, was discarded as well, as shown using also a deuterated analogue. The third suggested precursor of 3MH is a glutathionyl-3MH (G-3MH), which upon proteolytic degradation could release Cys-3MH. The knockout of the OPT1 gene, which encodes the major glutathione transporter, reduces 3MH accumulation by a 2-fold factor in grape must as compared to the wild type strain. Consequently, it is deduced that major 3MH precursor(s) are transported into yeast via Opt1p, which is in favor of G-3MH being a 3MH precursor. This work opens the search for the major natural precursor(s) of 3MH in Sauvignon Blanc must.     

Oral infection with a Shiga toxin-negative Escherichia coli O157:H7 strain elicits humoral and cellular responses but does not protect sheep from colonisation with the homologous strain

We have previously shown that rectally inoculated sheep excrete Escherichia coli O157:H7 during weeks to months without developing a clear antibody response. However, antibodies against this bacterium were observed in naturally infected sheep, which most likely became orally infected. To understand this difference, sheep were orally inoculated with the same Shiga toxin-negative E. coli O157:H7 strain that was used for the rectal inoculation. A primary oral inoculation resulted in shedding of E. coli O157:H7 in the faeces and detection of antibody responses against intimin, EspA and EspB. The antibody titres waned as shedding decreased. A secondary inoculation resulted in longer shedding, even though a booster antibody response occurred. Cellular responses followed a similar pattern as the antibody levels, albeit with a lower secondary response. The presence of antigen-specific antibody-secreting cells indicates involvement of both a systemic response in the spleen and a local immune response in the terminal rectum. These results suggest that E. coli O157:H7 has to pass the small intestine to evoke antibody responses.