Effect of feeding level and feeding frequency on specific dynamic action in Silurus meridionalis

The effect of feeding level ( F _L; 0·5 to 4% dry diet mass per wet fish body mass) and feeding frequency (once every 4 days to twice per day) on postprandial metabolic response was investigated in southern catfish Silurus meridionalis at 27·5° C. The results showed that there was no significant difference in the specific dynamic action (SDA) coefficient among the groups of different feeding levels ( P  > 0·05). The duration increased from 26·0 to 40·0 h and the peak metabolic rate increased from 207·8 to 378·8 mg O₂ kg⁻¹ h⁻¹ when the feeding level was increased from 0·5 to 4%. The relationship between the peak metabolic rate ( R _P, mg O₂ kg⁻¹ h⁻¹) and F _L could be described as: R _P = 175·4 + 47·3 F _L( r ² = 0·943, n  = 40, P  < 0·001). The relationship between the SDA duration ( D , h) and F _L could be described as D =30·97 F _L^0·248 ( r ²=0·729, n =40, P  < 0·001).     

Effects of temperature, body size and feeding on rates of metabolism in young‐of‐the‐year haddock

The mean rate of oxygen consumption (routine respiration rate, R _R, mg O₂ fish⁻¹ h⁻¹), measured for individual or small groups of haddock Melanogrammus aeglefinus (3–12 cm standard length, L _S) maintained for 5 days within flow‐through respiratory chambers at four different temperatures, increased with increasing dry mass ( M _D). The relationship between R _R and M _D was allometric ( R _R = α  M ^b ) with b values of 0·631, 0·606, 0·655 and 0·650 at 5·0, 8·0, 12·0 and 15·0° C, respectively. The effect of temperature ( T ) and M _D on mean R _R was described by     indicating a Q ₁₀ of 2·27 between 5 and 15° C. Juvenile haddock routine metabolic scope, calculated as the ratio of the mean of highest and lowest deciles of R _R measured in each chamber, significantly decreased with temperature such that the routine scope at 15° C was half that at 5° C. The cost of feeding ( R _SDA) was c . 3% of consumed food energy, a value half that found for larger gadoid juveniles and adults.     

Model experiments to establish behaviour of Yersinia enterocolitica O:9 strains in various types of fresh dry sausage

In model experiments different kinds of raw sausages were inoculated with liquid cultures of virulent-plasmid-carrying clinical Yersinia (Y.) enterocolitica (e.) strains of the O:9 serotype, doses being between 10⁴ and 10⁵ cfu g^-1. The sausage samples were stored at 3–5° and 13–16°C. During the first 10 d of storage the Y.e. plate count was detected with Desoxycholate-Citrate-Lactose-Sucrose Agar every day, later on in addition to it with phosphate buffer-enrichment and with enrichment according to Schiemann (1982) in intervals of several days' duration. The pH and a _w values, the contents of salt and water were detected. The multitude of complexly acting factors and substances prevents obviously the proliferation of Y.e. in fresh dry sausages. Decay dynamics of Y.e. were found to be considerably affected by storage temperature. Cold storage, basically, had a conservation effect and thus delayed the dying process of model strains. Yersinia enterocolitica -contaminated fresh dry sausage may cause potential danger to consumers, because of relatively extended survival periods of the pathogen. Therefore, manufacturers are expected to observe most stringent hygienic rules of Good Manufacturing Practice.     

Fate of low temperature and acid-adapted Yersinia enterocolitica and Listeria monocytogenes that contaminate lactic acid decontaminated meat during chill storage

Pathogens found in the environment of abattoirs may become adapted to lactic acid used to decontaminate meat. Such organisms are more acid tolerant than non-adapted parents and can contaminate meat after lactic acid decontamination (LAD). The fate of acid-adapted Yersinia enterocolitica and Listeria monocytogenes, inoculated on skin surface of pork bellies 2 h after LAD, was examined during chilled storage. LAD included dipping in 1%, 2% or 5% lactic acid solutions at 55°C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at ≈1–1·5 pH units below that of water-treated controls. Growth permitting pH at 4·8–5·2 was reached after 1% LAD in less than 0·5 d (pH 4·8–5·0), 2% LAD within 1·5 d (pH 4·9–5·1) and after 5% LAD (pH 5·0–5·2) within 4 d. During the lag on 2% LAD meat Y. enterocolitica counts decreased by 0·9 log₁₀ cfu per cm² and on 5% LAD the reduction was more than 1·4 log₁₀ cfu per cm². The reductions in L. monocytogenes were about a third of those in Y. enterocolitica . On 1% LAD the counts of both pathogens did not decrease significantly. The generation times of Y. enterocolitica and L. monocytogenes on 2–5% LAD meats were by up to twofold longer than on water-treated controls and on 1% LAD-treated meat they were similar to those on water-treated controls. Low temperature and acid-adapted L. monocytogenes and Y. enterocolitica that contaminate skin surface after hot 2–5% LAD did not cause an increased health hazard, although the number of Gram-negative spoilage organisms were drastically reduced by hot 2–5% LAD and intrinsic (lactic acid content, pH) conditions were created that may benefit the survival and the growth of acid-adapted organisms.     

Impact of temperature on food intake and growth in juvenile burbot

The effect of temperature on food consumption, food conversion and somatic growth was investigated with juvenile burbot Lota lota (age 0 years). Juvenile burbot showed a significant dome shaped relationship between relative daily food consumption ( C _R) and temperature ( T ) with C _R = − 0·00044 T ² + 0·01583 T  − 0·06010; ( n  = 90, r ² = 0·61). Maximum C _R was at 17·9° C (95% CL 17·2–18·6° C). The temperature related instantaneous growth rate ( G ) also followed a dome shaped function with G  = − 0·000063 T ² + 0·002010 T  − 0·007462; ( n  = 95, r ² = 0·57), with maximum growth rate at 16·0° C (95% CL 15·3–16·6° C). A significant linear relationship was found between the water temperature and the conversion coefficient ( C _C) with C _C = − 1·63 T  + 59·04; ( n  = 80, r ² = 0·74). The results indicate that juvenile burbot in large lakes benefit from higher water temperatures in the littoral zone, by increased food uptake and growth, especially during the warm summer months. Because profundal water temperatures do not reflect the optimal temperature for food consumption in large burbot, temperature is unlikely to be the main proximate factor for the obligate littoral‐profundal migration of juvenile burbot observed in many lake populations.     

Swimming performance, oxygen consumption and excess post-exercise oxygen consumption in adult transgenic and ocean-ranched coho salmon

Routine oxygen consumption ( M o ₂) was 35% higher in 1 day starved and 21% higher in 4 day starved adult transgenic coho salmon Oncorhynchus kisutch relative to end of migration ocean-ranched coho salmon. Critical swimming speed ( U _crit) and M o ₂ at U _crit ( M o _2max) were significantly lower in 4 day starved transgenic coho salmon (1·25 BL s⁻¹; 8·79 mg O₂ kg⁻¹ min⁻¹) compared to ocean-ranched coho salmon (1·60 BL s⁻¹; 9·87 mg O₂ kg⁻¹ min⁻¹). Transgenic fish swam energetically less efficiently than ocean-ranched fish, as indicated by a poorer swimming economy at U _crit ( M o _2max     ). Although M o _2max was lower in transgenic coho salmon, the excess post-exercise oxygen consumption (EPOC) measured during the first 20 min of recovery was significantly larger in transgenic coho salmon (44·1 mg O₂ kg⁻¹) compared with ocean-ranched coho salmon (34·2 mg O₂ kg⁻¹), which had a faster rate of recovery.     

Decreased time for detection and quantification of virulent Bacillus anthracis and Yersinia pestis using a BioNanoPore (BNPTM) membrane technology

Many aspects of biodefense research require quantitative growth assessments of the test agent. This study evaluated the BioNanoPore (BNP™) technology to quantitate Bacillus anthracis and Yersinia pestis faster than traditional plate counting methods. The BNP™ technology enabled quantification of B. anthracis and Y. pestis in phosphate-buffered saline and naïve rabbit blood at 6 and 24 h, respectively. After 6 h of growth, counts for B. anthracis ranged from 6·19–6·45 log₁₀ CFU ml⁻¹ on BNP™, while counts after 24 h on tryptic soy agar (TSA) ranged from 6·51–6·58 log₁₀ CFU ml⁻¹. For Y. pestis , counts on BNP™ at 24 h ranged from 6·31–6·41 log₁₀ CFU ml⁻¹ on BNP™ and ranged from 6·44–6·89 log₁₀ CFU ml⁻¹ on TSA at 48 h. This study demonstrates that the BNP™ technology provides a more rapid detection of B. anthracis and Y. pestis , which could aid in the evaluation of potential medical countermeasures and treatments as well as other biological defense applications such as surface sampling or decontamination efficacy.     

Isolation of Yersinia enterocolitica-resembling Organisms and Alteromonas putrefaciens from Vacuum-Packed Chilled Beef Cuts

Yersinia enterocolitica -resembling organisms were found at levels of 10⁷/g on a high pH (pH ≧ 6·0) vacuum-packaged beef striploin held for 6 weeks at 0·2°C, but did not exceed 10⁵/g on normal pH (pH < 6·0) striploins held for 10 weeks. Gram negative bacteria that produced H₂S on peptone iron agar were isolated from high pH vacuum packed striploins. These organisms were identified as Alteromonas putrefaciens . They attained levels of about 10⁷/g in 6 weeks at 0–2°C, at which time greening of the fat surface and 'drip'had occurred. On meat of normal pH, counts of A. putrefaciens were less than 10⁴/g after 6 weeks and no greening was evident.     

Inhibitory effects of sucrose fatty acid esters on survival of Yersinia enterocolitica on eggshell surface and use of blue lake as indicator of bacterial penetration into eggs

Aims:  To determine the effectiveness of sucrose monolaurate (SML) and sucrose monocaprate (SMC), alone and in combination with ethylenediaminetetraacetic acid (EDTA), propionic acid (PA) or citric acid (CA) in reducing mesophilic aerobic bacteria (MAB) and Yersinia enterocolitica O:9 populations on eggshells and their damage potential on the microstructure of shell cuticle.
Methods and Results:  Uninoculated eggs and eggs submerged in a solution of Y. enterocolitica were immersed in solutions of the various treatments. MAB and Y. enterocolitica counts on the surface of the eggs were carried out before and after treatment. MAB counts decreased less than 2 logs on uninoculated eggshells irrespective of treatment and reductions of 3·2 and 3·0 logs of Y. enterocolitica were obtained with 1000 μg ml⁻¹ SML plus 0·1% CA or 1000 μg ml⁻¹ SML plus 600 μg ml⁻¹ EDTA solutions, respectively. Y. enterocolitica 2/O:9 was recovered from natural microflora. Use of blue lake staining revealed minimal damage to the shells from the washing treatments.
Conclusions:  SML and SMC at 1000 μg ml⁻¹ combined with CA or EDTA could be effective in reducing Y. enterocolitica on eggshells with a minimal risk of later bacterial recontamination.
Significance and Impact of the Study:  Eggs are a recognized vehicle for transmission of Y enterocolitica although a prevalence of only 2·7% was detected in this study. Washing eggs in solutions containing SML or SMC could eliminate Y. enterocolitica contamination of egg shells.     

A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 10²–3·4 × 10³ cfu ml⁻¹ (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 10²–1·9 × 10⁴ cfu per microplant sample (equivalent to 5 cfu – 3·8 × 10² cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.     

Early growth and juvenile population structure of lemon sharks Negaprion brevirostris in the Atol das Rocas Biological Reserve, off north‐east Brazil

Lemon sharks Negaprion brevirostris were sampled in the Atol das Rocas, a nursery area, on nine occasions from March 1999 to October 2003, during which 157 individuals were tagged and 35 were recaptured. The male : female sex ratio of captured individuals was 1 : 1·12. Mean ±  s . d . growth rates were 24·7 ± 3·4 cm year⁻¹ in total length ( L _T), 20·7 ± 3·2 cm year⁻¹ in fork length, and 19·5 ± 2·7 cm year⁻¹ in precaudal length. There was no significant difference in growth rates between males and females. Mean ±  s . d . increase in mass was 2565 ± 762 g year⁻¹. The von Bertalanffy growth parameters estimated by the Fabens method based on L _T were: k  = 0·077, L _∞ = 399·9 cm and t ₀ = −2·16. Despite the large variation of environmental conditions, particularly of tidal range and currents, and the lack of protective mangrove cover in the nursery area at Atol das Rocas, juvenile lemon sharks grew relatively faster than at other nurseries. Such rapid growth could be a response to abundant food availability or high risk of predation by adults that enter the nursery area.     

Microbiological composition of raw milk from selected farms in the Camembert region of Normandy

Raw milk from 27 farms was sampled over 6 months for listerias, salmonellas, Yersinia enterocolitica and campylobacters. Total bacterial counts and somatic cell counts were measured. Lactococci, lactobacilli, dextran-producing leuconostocs, Brevibacterium linens , yeasts and moulds, Staphylococcus aureus and other Micrococcaceae, Pseudomonas , coliforms, Escherichia coli , enterococci, Clostridium perfringens and spores of anaerobic lactate-fermenting bacteria were also counted. Pseudomonas (2000 cfu ml⁻¹), lactococci (760 cfu ml⁻¹) and Micrococcaceae (720 cfu ml⁻¹) were the most numerous groups. Lactic acid bacteria were detected in all samples. Coliforms were present in most samples, but 84% of samples had counts <100 cfu ml⁻¹. Staphylococcus aureus was detected in 62% of milks, the average count was 410 cfu ml⁻¹. About 80% of supplies had ≤10 E. coli cfu ml⁻¹ and all samples had 1 Cl. perfringens cfu ml⁻¹. Two of the tested milks were positive for salmonellas (2·9%), four were positive for Listeria monocytogenes (5·8%), 25 for Yersinia enterocolitica (36%) and one for campylobacters (1·4%).     

Comparison of the Vitek, API 20E and Gene-trak® systems for the identification of Yersinia enterocolitica

The current improvements in nucleic acid hybridization technology provide new techniques for the identification of micro-organisms. One such technique is the Gene-trak® DNA hybridization system (Framingham, MA, USA), which was introduced in 1983. The objective for this study was to evaluate the new Gene-trak® Yersinia enterocolitica kit in comparison with the API 20E and Vitek systems. A total of 101 strains including 18 reference non- Yersinia strains from the authors' stock culture collection and 83 suspected positive isolates from CIN agar were tested. Of these 83 isolates, 40 were identified as Y. enterocolitica after incubation at 37°C for 24 with the API 20E system; 37 strains were identified at 30°C for 48 h. The Gene-trak® method gave positive results with 39 strains. The Vitek system gave positive results with 27 strains.
With the Gene-trak® method, Y. enterocolitica was detectable in mixed cultures provided that the numbers of cfu ml^-1 were equal to or above 10⁶ Y. enterocolitica ml^-1. Although enrichment procedures are still needed, the system provides a quick detection of these food-borne pathogens.     

Evaluation of PCR assays for quantifying seed-borne infection by Fusarium and Microdochium seedling blight pathogens

Aims:  To evaluate competitive PCR assays for quantifying seed-borne Microdochium and Fusarium seedling blight pathogen DNA and to determine test and year repeatability and sources of variability.
Methods and Results:  Relationships between DNA and plate counts were significant for Fusarium and Microdochium seedling blight pathogens in 152 seed batches from 3 years. Coefficient of determinations, however, differed greatly ( Fusarium ; R ² = 0·25, P  =   0·029, Microdochium ; R ² = 0·73, P  <   0·001). Significant differences between years were observed in the regression slopes for Microdochium . Pathogen DNA quantified in 16 extractions after sampling was highly correlated to results following storage for 1–2 years ( R ² > 0·90). Residual maximum likelihood analysis showed that the least and greatest variance components of the testing procedure were DNA extraction subsampling and PCR assay respectively.
Conclusions:  Amount of pathogen DNA is a useful estimator of seed batch contamination for Microdochium but not Fusarium seedling blight pathogens. Although reproducible over time, improvements to the testing procedure should focus on repeated PCR amplifications to reduce assay variability.
Significance and Impact of the Study:  Replacing plate counts with competitive PCR for determining the severity of seed batch contamination is feasible in areas where Microdochium seedling blight pathogens predominate.     

Structural stability of Sclerotium rolfsii ATCC 201126 β-glucan with fermentation time: a chemical, infrared spectroscopic and enzymatic approach

Aims:  Sclerotium rolfsii ATCC 201126 exopolysaccharides (EPSs) recovered at 48 h (EPS I) and 72 h (EPS II) of fermentation, with differences in rheological parameters, hydrogel topography, salt tolerance, antisyneresis, emulsifying and suspending properties, were subjected to a polyphasic characterization in order to detect structural divergences.
Methods and Results:  Fermenter-scale production led to productivity ( P _r) and yield ( Y _P/C) values higher at 48 h ( P _r = 0·542 g l⁻¹ h⁻¹; Y _P/C = 0·74) than at 72 h ( P _r = 0·336 g l⁻¹ h⁻¹; Y _P/C = 0·50). Both EPSs were neutral glucose-homopolysaccharides with a β-(1,3)-glycosidic backbone and single β-(1,6)-glucopyranosyl sidechains regularly attached every three residues in the main chain, as revealed by chemical analyses. The infra-red diagnostic peak at 890 cm⁻¹ confirmed β-glycosidic linkages, while gentiobiose released by β-(1,3)-glucanases confirmed single β-1,6-glycosidic branching for both EPSs.
Conclusions:  The true modular repeating unit of S. rolfsii ATCC 201126 scleroglucan could be resolved. Structural stability was corroborated and no structural differences could be detected as to account for the variations in EPSs behaviour.
Significance and Impact of the Study:  Recovery of S. rolfsii ATCC 201126 scleroglucan at 48 h might be considered based on better fermentation kinetic parameters and no detrimental effects on EPS structural features.     

The reproduction, age and growth of the spotted rockling

Biology of the Macaronesian endemic rockling Gaidropsarus guttatus was studied in the Azores. The overall sex ratio from the samples was highly in favour of females (1 : 6·33). The growth parameters were L _∞ = 24·23, k  = 1·219 and t ₀ = −0·059. Fish matured at 15 cm L _T and the spawning season was strongly clustered in April.     

Big and beautiful? Quantitative genetic parameters for appearance of large rainbow trout

A quantitative genetic analysis of body condition, body shape, skin colour and spottiness of large, farmed rainbow trout Oncorhynchus mykiss showed that the traits can be modified through selective breeding. This was indicated by their high heritabilities ( h ² = 0·46–0·61 on the underlying liability scale and 0·29–0·50 on the observed scale). Correlations calculated using linear models showed that skin colour and the amount of spots displayed positive phenotypic ( r _P = 0·33) and genetic correlations ( r _A = 0·83), the relationship being advantageous for the genetic improvement of the traits. Body shape and condition factor displayed disadvantageous correlations with body mass at both ages, the genetic correlations between the traits ranging from 0·36 to 0·57. It was concluded that there are no strong genetic constraints for the genetic improvement of appearance, the only limitation being that rapidly growing fish become rotund.     

Multiplex real-time PCR for exhaustive detection of diarrhoeagenic Escherichia coli

Aims:  The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC.
Methods and Results:  Primers and TaqMan probes were designed to amplify and quantify one gene ( eae , stx1 , stx2 , elt , est , virB , aggR , astA, and afaB ) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0·99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7·1 × 10² to 1·1 × 10⁴ CFU ml⁻¹, depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g⁻¹) were found to be positive by the method.
Conclusions:  The present system allows for the efficient and simultaneous determination of various DEC pathotypes.
Significance and Impact of the Study:  This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

Effect of selected antimicrobial compounds on the radiosensitization of Salmonella Typhi in ground beef

Aims:  In this study, we extended our previous work to determine the efficiency of antimicrobial compounds in increase of relative radiosensitivity of Salmonella Typhi in medium fat ground beef (23% fat) by testing 41 different essential oils (EOs), oleoresins and food sauces.
Methods and Results:  Ground beef samples inoculated with Salmonella Typhi (10⁶ CFU g⁻¹ ) were treated with each antimicrobial compound at a concentration of 0·5% (w/w). Then, the samples (25 g each) were packaged under air and irradiated in a ⁶⁰Co irradiator at doses from 0 to 1·75 kGy. Radiosensitivity was evaluated by calculating relative radiation sensitivity, defined as the ratio of radiation D ₁₀ value in the absence/presence of antimicrobial compound.
Conclusions:  Depending on the compound tested, the addition of antimicrobial compound decreased the D ₁₀ value of Salmonella Typhi, resulting in an increase of the radiation sensitivity up to more than four times. Among these antimicrobial compounds, Chinese cinnamon EO, clove EO and trans -cinnamaldehyde were most effective to increase the radiosensitivity of Salmonella Typhi in ground beef.
Significance and Impact of the Study:  These observations demonstrate that some active compounds can function as radiosensitizers of Salmonella Typhi.