Deirdre Gilroy

Fish feed as a source of oxytetracycline-resistant bacteria in the sediments under fish farms

Abstract Concentrations of oxytetracycline and the frequency of oxytetracycline resistance in the environmental microflora were monitored following the therapeutic use of this agent at a marine fish farm. 529 kg of oxytetracycline were administered over a 24 day period at an average of 1.4 kg per cage per day. Three days after the end of the therapy 4.6 ± 3.7 μg/g oxytetracycline were detected in the sediments and the frequency of resistance in the sediment microflora was 9.0 ± 5.3%. A rise in the frequency of resistance in this flora to 26 ± 8.7% occurred 24 days after the therapy. This rise was not associated with any increase in the concentrations of oxytetracycline in the sediment. At this time the frequency of resistance in the flora isolated from mussels suspended above the sediments (36 ± 8.5%) was significantly ( P = 0.005) higher than that present in the sediment flora. The feed used on the farm 24 days after the end of therapy was shown to contain 4.6 × 10 4 oxytetracycline-resistant cfu/g. The distribution of phenotypic groups in the oxytetracycline-resistant flora in this feed and in the sediments during the peak in resistance were compared with those from other marine environments. These data demonstrated that resistant flora in feed can, under certain circumstances, significantly contribute to the resistant flora detected in sediments under fish cages.

Spatial distribution of oxytetracycline and elevated frequencies of oxytetracycline resistance in sediments beneath a marine salmon farm following oxytetracycline therapy

Abstract The concentrations of oxytetracycline and the frequencies of oxytetracycline resistant microorganisms were determined in 11 samples taken from the sediments in the vicinity of a block of fish cages at a marine salmon farm. The cage block contained 10 tonnes of Atlantic salmon smolts and a total of 20 kg of oxytetracycline were administered during the 12 day treatment. Samples cores were collected by divers 5 days after the end of the period of therapy and the top 2 cm of each core was analysed. HPLC analysis was able to quantify the oxytetracycline concentrations in three of the six samples taken directly under the cage block. The mean concentration under the cage was between 0.65 and 1.2 μg g −1 ( n = 6) depending on the values attributed to samples where the concentrations were below the level of quantitation (1.2 μg g −1 ). In the five samples taken from locations not directly under the cage block oxytetracycline was only detected in the sample taken adjacent to, and down current from, the cage block. This sample was collected 10 m to the west of the cage block and contained 4.2 μg g −1 oxytetracycline. These data indicate that oxytetracycline was confined to an area of the sediment which was smaller in extent than the area of the cage block itself. The frequencies of resistance to oxytetracycline in the microflora cultured from the samples were determined by differential plating on 2216V media, containing 25 and 100 μg ml −1 oxytetracycline. Analysis of eighty three samples from sites free of overt human influence demonstrated that the background levels of resistance at these two selection concentrations were 1.3 ± 1.3% and 0.4 ± 0.6%, respectively. Elevated frequencies of resistance were detected in samples from a wider area than the cage block. The median frequency of resistance in the samples ( n = 6) taken from directly under the cage block was 1.4% at 100 μg ml −1 and 5.3% at 25 μg ml −1 . In the samples ( n = 5) taken from outside the cage block the frequencies were 5.3% at 100 μg ml −1 and 8.5% at 25 μg ml −1 . There was no correlation between the concentration of oxytetracycline in a sample and the frequency of resistance that was determined in the culturable microflora in that sample.

Genomic Comparisons of Salmonella enterica Serovar Dublin, Agona, and Typhimurium Strains Recently Isolated from Milk Filters and Bovine Samples from Ireland, Using a Salmonella Microarray

ABSTRACT Salmonella-induced enterocolitis is the leading food-borne illness with a lethal outcome and causes millions of cases of gastroenteritis each year. We examined genomic variation among 12 environmental, veterinary, and clinical Salmonella enterica serovar Dublin, Agona, and Typhimurium strains isolated in Ireland between 2000 and 2003, as well as two clinical isolates from Canada and four archival isolates, which belonged to serovars Dublin and Agona. Using DNA-DNA hybridization to a microarray consisting of most of the predicted protein-encoding sequences of the S. enterica serovar Typhimurium LT2 genome, we identified a number of genomic regions that were absent in one or more serovars. The 34 genomic regions encoded proteins involved in sugar metabolism, transport, fimbrial and phage biogenesis, and transcriptional regulation, as well as inner and outer membrane-associated proteins. Two of the four prophages identified in strain LT2, prophages Gifsy-1 and Gifsy-2, were present in all six serovar Typhimurium strains examined. Prophage Fels-1 was absent from all 18 isolates examined, and Fels-2 was completely absent from the serovar Typhimurium isolates and the Salmonella Reference Collection B serovar Dublin strain Du2. All five Salmonella pathogenicity islands were present in all isolates. Plasmid pSLT was absent from all serovar Agona isolates, and only homologues of the spv genes were present in eight of the nine serovar Dublin strains. Only limited intraserovar diversity was found among the nine serovar Dublin, three serovar Agona, and six serovar Typhimurium isolates examined even though these isolates had extensive geographic, temporal, and source differences.

Hawthorn (Crataegus spp.) in the treatment of cardiovascular disease.

The medicinal properties of hawthorn (Crataegus spp., a genus comprising approximately 300 species) have been utilized by many cultures for a variety of therapeutic purposes for many centuries. In the Western world cardiovascular disease (CVD) has become one of the single most significant causes of premature death. Echoing this situation, more recent research into the therapeutic benefits of hawthorn preparations has focused primarily upon its cardiovascular effects. This review covers research into the various mechanisms of action proposed for Crataegus preparations, clinical trials involving Crataegus preparations, and the herbs safety profile. Clinical trials reviewed have been inconsistent in terms of criteria used (sample size, preparation, dosage, etc) but have been largely consistent with regard to positive outcomes. An investigation into data available to date regarding hawthorn preparations and herb/drug interactions reveals that theoretical adverse interactions have not been experienced in practice. Further, adverse reactions relating to the use of hawthorn preparations are infrequent and mild, even at higher dosage ranges. A recent retrospective study by Zick et al. has suggested a negative outcome for the long-term use of hawthorn in the prognosis of heart failure. These findings are examined in this paper. Although further research is needed in certain areas, current research to date suggests that hawthorn may potentially represent a safe, effective, nontoxic agent in the treatment of CVD and ischemic heart disease (IHD).

Rapid Multiplex PCR and Real-Time TaqMan PCR Assays for Detection of Salmonella enterica and the Highly Virulent Serovars Choleraesuis and Paratyphi C

ABSTRACT Salmonella enterica is a human pathogen with over 2,500 serovars characterized. S. enterica serovars Choleraesuis and Paratyphi C are two globally distributed serovars. We have developed a rapid molecular-typing method to detect serovars Choleraesuis and Paratyphi C in food samples by using a comparative-genomics approach to identify regions unique to each serovar from the sequenced genomes. A Salmonella-specific primer pair based on oriC was designed as an internal control to establish accuracy, sensitivity, and reproducibility. Serovar-specific primer sets based on regions of difference between serovars Choleraesuis and Paratyphi C were designed for real-time PCR assays. Three primer sets were used to screen a collection of over 100 Salmonella strains, and both serovars Choleraesuis and Paratyphi C gave unique amplification patterns. To develop the technique for practical use, its sensitivity for detection of Salmonella spp. in a food matrix was determined by spiking experiments. The technique was also adapted for a real-time PCR rapid-detection assay for both serovars Choleraesuis and Paratyphi C that complements the current procedures for Salmonella sp. isolation and serotyping.

Influence of extraction technique on the anti-oxidative potential of hawthorn (Crataegus monogyna) extracts in bovine muscle homogenates

Six extracts were prepared from hawthorn (Crataegus monogyna) leaves and flowers (HLF) and berries (HB) using solid-liquid [traditional (T) (HLFT, HBT), sonicated (S) (HLFS, HBS)] and supercritical fluid (C) extraction (HLFC, HBC) techniques. The antioxidant activities of HLF and HB extracts were characterised using in vitro antioxidant assays (TPC, DPPH, FRAP) and in 25% bovine muscle (longissimus lumborum) homogenates (lipid oxidation (TBARS), oxymyoglobin (% of total myoglobin)) after 24h storage at 4°C. Hawthorn extracts exhibited varying degrees of antioxidant potency. In vitro and muscle homogenate (TBARS) antioxidant activity followed the order: HLFS>HLFT and HBT>HBS. In supercritical fluid extracts, HLFC>HBC (in vitro antioxidant activity) and HLFC≈HBC (TBARS). All extracts (except HBS) reduced oxymyoglobin oxidation. The HLFS extract had the highest antioxidant activity in all test systems. Supercritical fluid extraction (SFE) exhibited potential as a technique for the manufacture of functional ingredients (antioxidants) from hawthorn for use in muscle foods.

Confusion generated by the validation of the application of an enzyme linked immunosorbent assay and a polymerase chain reaction method for the detection of Aeromonas salmonicida in field samples

Abstract Two protocols designed to detect Aeromonas salmonicida, one based on an enzyme linked immunosorbent assay (ELISA) and one on a polymerase chain reaction (PCR), had previously performed well in extensive laboratory-based validation studies. These proxy methods were used together with direct culture to examine 223 kidneys of wild salmonid fish and 35 sediment samples collected from their habitat. Direct culture failed to detect the organism in any of the kidney samples. ELISA generated positive results from 23.3% of the kidneys and PCR from 20.6% but the degree of agreement between the two proxy methods was not greater than would have been predicted by chance (p>0.05). A total of 60% of the samples generating a positive response in ELISA were negative by PCR and 56% of PCR-positive samples were negative by ELISA. The overall concordance was, therefore, only 27%. With respect to the sediment samples, ELISA generated positive results from 53% and PCR from 33%. Again, the degree of agreement between the two proxy methods was not significantly greater than chance and the concordance was only 24%. The data generated by the two proxy methods investigated in this work was employed in a comparative validation of their application to the detection of A. salmonicida in these sample matrices. The hypothesis that both the PCR and ELISA protocols used in this work can validly be used in this way is clearly inconsistent with the data obtained. This analysis could not, however, discriminate between the hypotheses that neither of the methods possessed validity for these applications or that one of them possesses considerably greater validity than the other. The extensive laboratory-scale validation studies to which the two proxy methods used in this work have been submitted are discussed. It is argued that these laboratory studies provided no grounds for predicting the lack of validity that was detected when these proxy methods were applied to field samples. The results presented in this work provide strong evidence that laboratory-scale studies are not adequate to validate the application of proxy methods to field samples.

Application-dependent, laboratory-based validation of an enzyme-linked immunosorbent assay for Aeromonas salmonicida

Abstract A heterologous, noncompetitive, sandwich, enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of Aeromonas salmonicida. This paper reports the initial, laboratory validation of the applications of this assay to the study of the ecology of A. salmonicida in the effluents of freshwater hatcheries, marine sediments and in fish. The selectivity of the assay was investigated by the use of four (one core and three application-dependent) control panels of bacteria. No false-negative and two false-positive reactions were detected in the core panel that contained 108 strains of the target species, 100 strains of nontarget mesophilic aeromonads and 19 miscellaneous strains. The two false-positive reactions were generated by strains of Staphylococcus aureus . In the 74-strain panel for application to fish tissues and the 150 strain panel for freshwater sediment applications, no false positives were detected. Two false positives were, however, detected in the 150-strain panel for applications to marine sediments. Nine samples of environmental matrices, which the proposed applications of this assay might require to be examined, were used in an investigation of performance characteristics of the assay in spiked and unspiked matrices. The matrices exerted a slight influence of the baseline values generated by the assay and reduced the intra- and interassay precision; however, in all cases, these changes were within acceptable limits. The applied lower detection limits for A. salmonicida in spiked environmental matrices were in the range 1–4×10 3 cfu/ml and were slightly higher than the limits detected in broth suspensions of the organism. The ELISA was also demonstrated to have the ability to detect A. salmonicida in studies of incurred freshwater and sediments. The laboratory experiments reported here failed to detect any evidence that would argue against the initiation of full-scale validation studies of this method under field conditions.

Furunculosis-inducing ability of microcosms seeded with Aeromonas salmonicida correlates with colony-forming ability but not with PCR and ELISA data

Two laboratory microcosms, constructed of sterilised sediment and water taken from different locations, were seeded with Aeromonas salmonicida. A non-quantitative PCR, a heterogeneous, non-competitive, sandwich ELISA and colony formation were employed to generate survival data. In one microcosm, all three analytical methods generated positive results over the full 279 days of the incubation of the system at 18 °C. In the second microcosm, constructed with humic acid-rich material, colony formation was undetectable after 1 day but the PCR and ELISA methods generated positive results for up to 269 days. Direct injection into fish of material from the two microcosms both seeded with a passaged strain of A. salmonicida was used to determine the disease-producing potential of the bacteria in these systems. A direct correlation was observed between the presence of colony-forming units and the ability to produce furunculosis in test fish. None of the samples that generated positive PCR and ELISA signals but in which colony formation was not detected proved capable of inducing disease in fish. The significance of these data for the application of proxy methods in epizootiological studies is discussed.