The cost of resistance to colistin in Acinetobacter baumannii: a proteomic perspective

Colistin resistance in Acinetobacter baumannii, a pathogen of clinical concern, was induced in the susceptible strain ATCC 19606 by growth under increasing pressure of the antibiotic, the only drug universally active against multi‐resistant clinical strains. In 2‐D difference gel electrophoresis (DIGE) experiments, 35 proteins with differences in expression between both phenotypes were identified, most of them appearing as down regulated in the colistin‐resistant strain. These include outer membrane (OM) proteins, chaperones, protein biosynthesis factors, and metabolic enzymes, all suggesting substantial loss of biological fitness in the resistant phenotype, as substantiated by complementary experiments in the absence of colistin. Results shed light on the scarcity of widespread clinical outbreaks for resistant phenotypes.     

Comparison of disc diffusion, Etest and agar dilution for susceptibility testing of colistin against Enterobacteriaceae

Aims: In this study, we compared different methods of colistin susceptibility testing, disc diffusion, agar dilution and Etest using a set of Enterobacteriaceae isolates that included colistin‐resistant strains. Methods and results: Susceptibility of 200 clinical isolates of Enterobacteriaceae to colistin was tested to compare agar dilution (reference method), disc diffusion (50 and 10 μg) and Etest. MICs (minimum inhibitory concentrations) were interpreted using the criteria established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Colistin exhibited excellent activity against Escherichia coli and E. cloacae (MIC₉₀ = 0·5 mg l^?1). In contrast, colistin was less active against Klebsiella pneumoniae (MIC₉₀ = 16 mg l^?1). Resistance rates varied from 0% in E. coli to 1·8% in E. cloacae and 13% in K. pneumoniae. High rates of very major errors were observed in the disc diffusion test using either the criteria of the Comité de l’antibiogramme de la Société Française de Microbiologie (CA‐SFM) or the criteria of the Clinical and Laboratory Standards Institute (CLSI), respectively, 3·5 and 2·5%. When the criteria of Gales et al. were applied, the number of very major errors was reduced to one (0·5%). The Etest showed good concordance with agar dilution method. Conclusion: Disc susceptibility testing methods are unreliable on detecting colistin resistance. MIC should be determined to confirm the susceptibility results by disc diffusion. Significance and Impact of the study: We recommend the determination of MIC by Etest for all multidrug‐resistant Enterobacteriaceae when colistin is required for the treatment.     

Effect of incubation temperature on muscle growth of barramundi Lates calcarifer at hatch and post-exogenous feeding

Muscle morphology was investigated in newly hatched barramundi Lates calcarifer larvae incubated at set temperatures (26, 29 and 31° C) prior to hatching. Three days after hatching (the start of exogenous feeding), larvae from the 26 and 31° C treatments were each divided into two groups and reared at that temperature or transferred over the period of several hours to 29° C (control temperature). Incubation temperature significantly affected muscle cellularity in the developing embryo, with larvae incubated at 26° C (mean ±s .e . 223·3 ± 7·9) having on average 14·4% more inner muscle fibres than those incubated at 31° C (195·2 ± 8·8) and 4·8% more than those incubated at 29° C (213·5 ± 4·7). Conversely, inner muscle fibre cross‐sectional area significantly increased at the warm incubation temperature in L. calcarifer, so that the total cross‐sectional muscle area was not different between treatment groups. The total cross‐sectional area of superficial muscle fibres and the proportion of superficial to total fibre cross‐sectional area in just hatched L. calcarifer were also affected by incubation temperature, with incubation at the cool temperature (26° C) increasing both the total cross‐sectional area and proportion of superficial muscle fibres. By 9 days post‐hatch, the aforementioned differences were no longer significant. Similarly, there was no difference in total superficial fibre cross‐sectional area between any treatment groups of L. calcarifer, whereas incubation temperature still significantly affected the proportion of superficial to total muscle fibre cross‐sectional area. Larvae hatched and grown at 31° C had a significantly reduced percentage of superficial muscle cross‐sectional area (mean ±s .e . 5·11 ± 0·66%) compared with those incubated and grown at 29° C (8·04 ± 0·77%) and 26° C (9·32 ± 0·56%) and those incubated at 26° C and transferred to 29° C (7·52 ± 0·53%), and incubated at 31° C and transferred to 29° C (6·28 ± 0·69%). These results indicate that changes in muscle cellularity induced by raising or lowering the incubation temperature of L. calcarifer display varying degrees of persistence over developmental time. The significance of these findings to the culture of L. calcarifer is discussed.     

Upper thermal tolerance of closely related Danio species

The main finding of this study was that measuring maximum heart rate during incremental warming was an effective tool to estimate upper thermal limits in three small cyprinid Danio species, which differed significantly. Arrhenius breakpoint temperature for maximum heart rate, purportedly an index of optimum temperature, was 21·2 ± 0·4, 20·1 ± 0·4 and 18·9 ± 0·8° C (mean ± s.e .) for zebrafish Danio rerio, pearl danio Danio albolineatus and glowlight danio Danio choprae, respectively. The temperature where cardiac arrhythmias were first induced during warming (T_arr) was 36·6 ± 0·7, 36·9 ± 0·8 and 33·2 ± 0·8° C (mean ± s.e .) and critical thermal maximum (T_Cm) was 39·9 ± 0·1, 38·9 ± 0·1 and 37·2 ± 0·1° C (mean ± s.e .) for D. rerio, D. albolineatus and D. choprae, respectively. The finding that T_arr was consistently 3–4° C lower than T_Cm suggests that collapse of the cardiac life support system may be a critical trigger for upper temperature tolerance. The upper thermal limits established here, which correlate well with a broad natural environmental temperature range for D. rerio and a narrow one for D. choprae, suggest that upper thermal tolerance may be a genetic trait even among closely related species acclimated to common temperatures.     

A finite interval of initial swimbladder inflation in Latris lineata revealed by sequential removal of water‐surface films

A finite interval of initial swimbladder inflation in striped trumpeter Latris lineata larvae occurred over 4 days at 16° C. Water‐surface films were removed on different days to form treatments: 4, 8, 9, 10, 11 and 12 days post hatching, dph (day 4, 8, 9, 10, 11 and 12 treatments, respectively). No swimbladder inflation was recorded prior to water‐surface film removal. When the water‐surface films were removed in day 4 and 8 treatments, initial swimbladder inflation was first recorded in larvae 9 dph at mean ± s .e . 35·0 ± 5·4%(n = 4) and 45·0 ± 7·9%, respectively. Water‐surface film removal at days 9, 10 and 11, resulted in initial swimbladder inflation the following day at 62·5 ± 2·5, 62·5 ± 7·2 and 11·3 ± 5·5% in larvae 10, 11 and 12 dph, respectively. No swimbladder inflation was recorded following water‐surface film removal on day 12. There was no significant difference in initial inflation among larvae in day 4, 8, 9 and 10 treatments, ranging from 65·0 ± 4·1 to 73·8 ± 6·9%(P > 0·05). Initial inflation was significantly lower in the day 11 treatment (11·3 ± 5·5%)(P < 0·05). During the inflation interval (9–12 dph) swimbladders displayed one of three morphologies; liquid dilation, gas inflated and collapsed. Collapse of the swimbladder lumen was first apparent in larvae without swimbladder inflation from 11 dph and progressively developed thereafter in all larvae with non‐inflated swimbladders. Larvae >6·1 mm standard length lost the ability to undergo initial swimbladder inflation. This study demonstrates that the interval for initial swimbladder inflation in striped trumpeter is short, finite and related to larval size. The end of the inflation interval was marked by onset of abnormal swimbladder morphologies, but not to closure of the pneumatic duct.     

Effects of holding temperature and handling stress on the upper thermal tolerance of threadfin shad Dorosoma petenense

The critical thermal maxima (T_MAX) of threadfin shad Dorosoma petenense exposed to standardized stress (30 s handling in a dip‐net), simulating stressors endured during fish loading before transport, were measured over a range of holding temperatures (15, 20 and 25° C). Dorosoma petenense T_MAX showed a significant thermal effect, displaying mean ±s.d . critical thermal maxima of 26·5 ± 1·6, 30·9 ± 1·2 and 33·3 ± 1·4° C, when tested at temperatures of 15, 20 and 25° C, respectively. Dorosoma petenense T_MAX levels were also affected by stress, with handled fish showing significantly lower values than control fish exposed to 15 (mean ±s.d . T_MAX = 25·6 ± 2·0° C), 20 (27·6 ± 2·8° C) and 25° C (32·0 ± 2·6° C). In addition to providing basic information on D. petenense thermal tolerance, experimental results suggest that fishery managers should consider the whole suite of potential stressors, such as air exposure during handling and fish loading, when developing management criteria.     

Long‐term effects of photoperiod,temperature and their interaction on growth,gill Na+, K+‐ATPase activity,seawater tolerance and plasma growth‐hormone levels in Atlantic salmon Salmo salar

This study was undertaken to examine the long‐term effects of photoperiod, temperature and their interaction on growth, gill Na⁺,K⁺‐ATPase (NKA) activity, seawater tolerance and plasma growth‐hormone levels in Atlantic salmon Salmo salar pre‐smolts and smolts. The fish (mean ± s.e . initial body mass = 15·9 ± 0·4 g) were reared on two photoperiods (continuous light, LL, and simulated natural photoperiod, LDN, 60° 25′ N) and two temperatures (8·3 and 12·7° C) from June to May of the following year. Mean body mass was affected by photoperiod, temperature and their interactions. Both temperature groups on LL developed peak levels in gill NKA activity from October to November, 4–5 months prior to the natural season for the parr–smolt transformation. Fish at 12° C showed peak levels in NKA activity 4–6 weeks before the fish at 8° C. Fish in all four experimental groups showed maximum NKA activity within a similar size range (113–162 g). The present findings further indicate that smoltification in S. salar is to some extent driven by size, and that S. salar will develop smolt characteristics, e.g. a marked increase in NKA activity, within a similar size range. Faster‐growing S. salar will, thus, reach this size threshold at a relatively younger age.     

Anti-biofilm activity of Quercus infectoria G. Olivier against methicillin-resistant Staphylococcus aureus

Aims: To establish the effect of Quercus infectoria G. Olivier extract and its main constituent, tannic acid, on staphylococcal biofilm and their anti‐biofilm mechanisms. Methods and Results: Anti‐biofilm activity of the plant materials on clinical isolated of methicillin‐resistant Staphylococcus aureus and methicillin‐susceptible Staph. aureus was employed using a crystal violet‐stained microtiter plate method. The extract at minimum inhibitory concentration (MIC; 0·25 mg ml^?1) was significantly reduced the biofilm formation of the isolates (P < 0·05). The effect on staphylococcal cell surface hydrophobicity (CSH) of the test compounds was investigated as a possible mode of action of the anti‐biofilm activity. The hydrophobicity index of all the bacterial isolates increased following treatment with supra‐MIC, MIC and sub‐MIC of the extract and tannic acid. Observation of the treated bacterial cells by electron microscopy revealed that the test compounds caused clumps of partly divided cocci with thickened and slightly rough cell wall. Conclusions: The results indicated that Q. infectoria extract and tannic acid affected staphylococcal biofilm formation and their effect on bacterial CSH and cell wall may involve in the anti‐biofilm activity. Significance and Impact of the Study: This evidence highlighted the anti‐biofilm potency of the natural products and clarified their anti‐biofilm mechanisms.     

The efficacy of the aquatic anaesthetic AQUI‐S® for anaesthesia of a small freshwater fish,Melanotaenia australis

For stage four anaesthesia of the western rainbowfish Melanotaenia australis the most efficacious concentration of AQUI‐S^® was found to be 80 mg l^?1 when five different doses were tested at 27° C (range 26·5–27·5° C). This gave a mean ±s.e. (n = 10) induction time of 140·4 ± 8·9 s and recovery time of 180·2 ± 8·9 s and resulted in 10% mortality when fish were continuously exposed for 15 min. All fish given this dose during induction and recovery time trials ate within 1 h of recovery, and there was zero mortality during a subsequent 1 month monitoring period.     

A new experimental model of acute osteomyelitis due to methicillin-resistant Staphylococcus aureus in rabbit

Aims: To assess the impact of antibiotic therapy on severe osseous infections, animal models of chronic bacterial infections have been developed; however, these models suffer from many experimental limitations. The aim of this work was to develop a new model system in which high levels of bacteria are obtained within femoral bone marrow and bone tissue, and such infections are maintained for at least 14 days. Methods and Results: Experimental osteomyelitis was induced in 25 New Zealand white rabbits. A 10⁹ CFU ml^?1 suspension of methicillin‐resistant Staphylococcus aureus was injected into the knee after bone trepanation. On day 3, surgical debridement was performed to mimic a surgical procedure. Animals were euthanized 1, 2, 3, 9 and 14 days post‐inoculation to determine the bacterial counts in marrow and bone, and to evaluate the stability of the infection. Inoculated lesions also were assessed for changes in histological parameters on days 3 and 7 post‐inoculation. At days 1, 2, 3, 9 and 14 post‐inoculation, we observed 6·50 ± 0·64, 7·30 ± 0·49, 7·82 ± 0·19, 8·00 ± 1·48 and 8·99 ± 0·20 log10 CFU g^?1 in bone marrow and 8·40 ± 0·68, 7·65 ± 0·27, 7·58 ± 0·30, 8·88 ± 0·52 and 8·28 ± 0·39 log10 CFU g^?1 in bone tissue, respectively. No statistical differences in bacterial count were found between bone marrow and bone tissue at any time point. Conclusion: This new model of acute osteomyelitis was validated by histological and microbiological changes in the absence of sclerosing agents, and these changes remained stable for 14 days. Significance and Impact of the Study: These results describe a new experimental model of acute osteomyelitis and demonstrate its usefulness in assessing the activity of antibacterial agents in vivo soon after bone infection.     

A rapid MALDI-TOF mass spectrometry-based method for colistin susceptibility testing in Escherichia coli

Colistin is recognized as a last-resort treatment option against multi-drug resistant bacteria including carbapenem-resistant Enterobacteriaceae (CRE). However, the plasmid-mediated colistin-resistance gene mcr-1 has been reported globally resulting in an increase of colistin-resistant bacteria. A quick and accurate method for determining the pathogen resistance of colistin is therefore crucial in the clinic. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a potential tool forto be applied for antimicrobial susceptibility testing. We compared the growth of Escherichia coli strains in the presence or absence of colistin. Automated analyses of the spectra were performed with a prototype software tool written with package R. Three mcr-1-positive and six mcr-1-negative E. coli were used for establishing the model to obtain the optimal incubation time, the breakpoint concentration of colistin and cut-off of the relative growth (RG) value. The distinction between susceptible and resistant strains was already noticeable after 2 h of incubation. The best separation between the susceptible and resistant strains was achieved at a concentration of 4 µg ml^-1 and a relative growth cut-off value of 0.6. Application of the model for the analysis of 128 E. coli isolates, a sensitivity of 97.4% and a specificity of 88.2% were achieved compared with colistin MIC results. The rapid MALDI-TOF MS-based method approach is simple to set-up, uses a short incubation time, and had excellent outcomes with respect to sensitivity and specificity for colistin sensitivity testing in Escherichia coli.     

Effect of acclimation temperature on the upper thermal tolerance of Colorado River cutthroat trout Oncorhynchus clarkii pleuriticus: thermal limits of a North American salmonid

In an effort to explore the thermal limitations of Colorado River cutthroat trout Oncorhynchus clarkii pleuriticus, the critical thermal maxima (T_cmax) of 1+ year Lake Nanita strain O. c. pleuriticus were evaluated when acclimated to 10, 15 and 20° C. The mean ±s.d. T_cmax for O. c. pleuriticus acclimated to 10° C was 24·6 ± 2·0°C (n = 30), for 15° C‐acclimated fish was 26·9 ± 1·5° C (n = 23) and for 20° C‐acclimated fish was 29·4 ± 1·1° C (n = 28); these results showed a marked thermal acclimation effect (Q₁₀ = 1·20). Interestingly, there was a size effect within treatments, wherein the T_cmax of larger fish was significantly lower than that of smaller fish acclimated to the same temperature. The critical thermal tolerances of age 0 year O. c. pleuriticus were also evaluated from three separate populations: Lake Nanita, Trapper Creek and Carr Creek reared under ‘common‐garden’ conditions prior to thermal acclimation. The Trapper Creek population had significantly warmer T_cmax than the Lake Nanita population, but that of the Carr Creek fish had T_cmax similar to both Trapper Creek and Lake Nanita fish. A comparison of these O. c. pleuriticus T_cmax results with those of other stream‐dwelling salmonids suggested that O. c. pleuriticus are less resistant to rapid thermal fluctuations when acclimated to cold temperatures, but can tolerate similar temperatures when acclimated to warmer temperatures.     

Life history of an unusual marine fish: survival, growth and movement patterns of Hippocampus guttulatus Cuvier 1829

The life history of the long‐snouted seahorse Hippocampus guttulatus was characterized using mark‐recapture data collected within a focal study site and catch data from 53 additional sites in the Ria Formosa coastal lagoon, southern Portugal. Population structure in benthic habitats was characterized by high local densities (0·3–1·5 m^?2), equal sex ratios and few juveniles <70 mm. Adult H. guttulatus maintained small (19·9 ± 12·4 m²), strongly overlapping home ranges during multiple reproductive seasons. Recruited (benthic) juveniles exhibited significantly lower site fidelity than adults. A Ford‐Walford plot of standard length (L_S) at time t against L_S measured during the previous year from tagged juveniles and adults led to estimates of the von Bertalanffy parameters K = 0·571 and L_∞ = 197·6 mm. The growth rate of planktonic juveniles (inferred from previous studies), was greater than predicted by the von Bertalanffy model, providing evidence of an ontogenetic shift in growth trajectory. The instantaneous rate of natural mortality, M, ranged from 1·13 to 1·22 year^?1(annual survival rate = 29·4–32·2%). Sexes did not differ in movement, growth or survival patterns. On average, H. guttulatus measured 12·2 ± 0·8 mm at birth. Planktonic juveniles recruited to vegetated habitat at 96·0 ± 8·0 mm (0·25 years), had mature brood pouches (males only) at 109·4 mm (0·49 years), began maintaining home ranges and reproducing at 125–129 mm (0·85–0·94 years), and lived for 4·3–5·5 years. Early age at maturity, rapid growth rates, and short generation times suggested that H. guttulatus may recover rapidly when direct (e.g. exploitation) and indirect (e.g. by‐catch and habitat damage) effects of disturbance cease, but may be vulnerable to extended periods of poor recruitment.     

Upper thermal limits of cardiac function for Arctic cod Boreogadus saida,a key food web fish species in the Arctic Ocean

The objective of this study was to determine the upper thermal limits of Arctic cod Boreogadus saida by measuring the response of maximum heart rate (f_Hmax) to acute warming. One set of fish were tested in a field laboratory in Cambridge Bay (CB), Nunavut (north of the Arctic Circle), and a second set were tested after air transport to and 6 month temperature acclimation at the Vancouver Aquarium (VA) laboratory. In both sets of tests, with B. saida acclimated to 0° C, f_Hmax increased during acute warming up to temperatures considerably higher than the acclimation temperature and the near‐freezing Arctic temperatures in which they are routinely found. Indeed, f_Hmax increased steadily between 0·5 and 5·5° C, with no significant difference between the CB and VA tests (P > 0·05) and with an overall mean ± s.e. Q₁₀ of 2·4 ± 0·5. The first Arrhenius breakpoint temperature (T_AB) for f_Hmax was also statistically indistinguishable for the two sets of tests (mean ± s.e. 3·2 ± 0·3 and 3·6 ± 0·3° C), suggesting that the temperature optimum for B. saida could be reliably measured after live transport to a more southerly laboratory location. Continued warming above 5·5° C revealed a large variability among individuals in the upper thermal limits that triggered cardiac arrhythmia (T_arr), ranging from 10·2 to 15·2° C with mean ± s.e. 12·4 ± 0·4° C (n = 11) for the field study. A difference did exist between the CB and VA breakpoint temperatures when the Q₁₀ value decreased below 2 (the Q₁₀ breakpoint temperature; T_QB) at 8·0 and 5·5° C, respectively. These results suggest that factors, other than thermal tolerance and associated cardiac performance, may influence the realized distribution of B. saida within the Arctic Circle.     

Changes in growth and maturation parameters of Pacific sardine Sardinops sagax collected off California during a period of stock recovery from 1994 to 2010

Whether fluctuation in density influenced the growth and maturation variables of three aggregated cohorts (fish born during the 1986–1993, 1996–2003 and 2004–2008 periods) of Pacific sardine Sardinops sagax caeruleus collected off the Californian coast from 2004 to 2010 was investigated. Using a von Bertalanffy mixed‐effects model with aggregated cohorts as covariates, estimated growth rate significantly covaried with aggregated cohorts. Growth rate (K) was modelled as a fixed effect and estimated to be 0·264 ± 0·015 (±s.e ). Statistical contrasts among aggregated cohorts showed that the 1996–2003 cohorts had a significantly lower growth rate than the other two aggregated cohorts. The theoretical age at length zero (t₀) and the standard length at infinity (L_S∞) were modelled as random effects, and were estimated to be ?2·885 ± 0·259 (±s.e ) and 273·13 ± 6·533 mm (±s.e ). The relation of ovary‐free mass at length was significantly different among the three aggregated cohorts, with the allometric coefficient estimated to be 2·850 ± 0·013 (±s.e ) for the S. sagax population. The age‐at‐length trajectory of S. sagax born between 1986 and 2008 showed strong density dependence effects on somatic growth rates. In contrast to the density‐dependent nature of growth, the probability to be mature at‐size or at‐age was not significantly affected by aggregated cohort density. The size and the age‐at‐50% maturity were estimated to be 150·92 mm and 0·56 years, respectively. Stock migration, natural fluctuations in biomass and removal of older and larger S. sagax by fishing might have been interplaying factors controlling growth parameters during 1986–2010.     

The effect of spine postures on the hydrodynamic drag in Epinephelus ongus larvae

Laboratory behavioural observation and computational fluid dynamics (CFD) analysis were conducted to examine whether the movement of the elongated dorsal and pelvic spines changed the hydrodynamic drag in white‐streaked grouper Epinephelus ongus larvae. The behavioural observation in the tank revealed that the larvae extended the dorsal and pelvic spines during passive transport and retracted during swimming; the angles of the dorsal and pelvic spines in relation to the anteroposterior axis were larger during the passive transport (mean ± s.d . = 28·84 ± 14·27 and 20·35 ± 15·05°) than those during the swimming (mean ± s.d . = 2·59 ± 5·55 and 0·32 ± 6·49°). The CFD analysis indicated that the relative hydrodynamic drag acting on the larvae was approximately 1·25 times higher when the spines were extended (passive transport) than when the spines were retracted (swimming), suggesting that the E. ongus larvae have an ability to adjust their hydrodynamic drag depending on the behavioural context.     

Formulation development of the biocontrol agent Bacillus subtilis strain CPA-8 by spray-drying

Aims: To prepare commercially acceptable formulations of Bacillus subtilis CPA‐8 by spray‐drying with long storage life and retained efficacy to control peach and nectarine brown rot caused by Monilinia spp. Methods and Results: CPA‐8 24‐h‐ and 72‐h‐old cultures were spray dried using 10% skimmed milk, 10% skimmed milk plus 10% MgSO₄, 10% MgSO₄ and 20% MgSO₄ as carriers/protectants. All carriers/protectants gave good percentages of powder recovery (28–38%) and moisture content (7–13%). CPA‐8 survival varied considerably among spray‐dried 24‐h‐ and 72‐h‐old cultures. Seventy‐two hours culture spray dried formulations showed the highest survival (28–32%) with final concentration products of 1·6–3·3 × 10⁹ CFU g^?1, while viability of 24‐h‐old formulations was lower than 1%. Spray‐dried 72‐h‐old formulations were selected to subsequent evaluation. Rehydration of cells with water provided a good recovery of CPA‐8 dried cells, similar to other complex rehydration media tested. Spray‐dried formulations stored at 4 ± 1 and 20 ± 1°C showed good shelf life during 6 months, and viability was maintained or slightly decreased by 0·2–0·3‐log. CPA‐8 formulations after 4‐ and 6 months storage were effective in controlling brown rot caused by Monilinia spp. on nectarines and peaches resulting in a 90–100% reduction in disease incidence. Conclusions: Stable and effective formulations of biocontrol agent B. subtilis CPA‐8 could be obtained by spray‐drying. Significance and Impact of the Study: New shelf‐stable and effective formulations of a biocontrol agent have been obtained by spray‐drying to control brown rot on peach.     

Growth, sex differentiation and gonad and plasma levels of sex steroids in male‐ and female‐dominant populations of Dicentrarchus labrax obtained through repeated size grading

Starting from 66 days post hatching (dph), European sea bass Dicentrarchus labrax were graded successively to create a fast growing (L‐extreme) and a slow growing (S‐extreme) population. The L‐extreme population grew significantly larger (ANOVA, n = 89–101, P < 0·01) attaining twice the wet mass of the S‐extreme population at 300 dph (130·9 ± 1·8 v. 66·7 ± 0·9 g, mean ± s .e .). When the two populations were sexed, the L‐extreme consisted of 96·5% and the S‐extreme of 30·2% females, while the ungraded control had 59·2% females. Sex differentiation began first in females at a total length (L_T) of 97 ± 4 mm and wet mass of 9·4 ± 1·2 g (150 dph), and was completed when fish reached 166 ± 6 mm and 53·4 ± 6·4 g (250 dph) in both sexes. Precocious maturation in males was positively correlated to growth. Gonad oestradiol (E₂) was significantly higher in the female‐dominant population at the onset of ovarian differentiation (ANOVA, n = 10, P < 0·05) and in the plasma after the appearance of the first primary oocytes (P < 0·01). Gonad testosterone (T) increased in both populations after sex differentiation (ANOVA, n = 10, P < 0·05), while plasma levels were significantly higher in the male‐dominant population (P < 0·001). Both gonad and plasma 11‐keto testosterone (11‐KT) were significantly higher in the male‐dominant population (ANOVA, n = 10, P < 0·01) reaching maximal values at spermiation. The results suggest that E₂ is closely related with ovarian differentiation and the onset of oogenesis, while T and 11‐KT is more related to spermatogenesis and precocious maturation.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

Prevention of the accumulation of Alicyclobacillus in apple concentrate by restricting the continuous process running time

Aims: To study the accumulation of vegetative cells and endospores of Alicyclobacillus, as well as viable aerobic counts during the continuous production of apple juice concentrate. Methods and Results: Apples were processed for a continuous process running time of 108 h (processing rate 1·8–2·0 t h^?1) without clean‐in‐place (CIP) procedures in‐between different batches. Samples from single‐strength apple juice, concentrate after evaporation (±30°Brix), the final product (concentrate pasteurized at 102–104°C for 90 s) and condensate water (by‐product of the juice concentration process) were collected every 12 h. From 12 to 84 h of processing, vegetative Alicyclobacillus counts in single‐strength apple juice increased significantly (P < 0·05) from 1 to 3·15 log₁₀ CFU ml^?1. Accumulation patterns of vegetative cells in apple concentrate and the final product were similar from 24 to 84 h of processing, with the respective counts increasing from 0·13 to 1·63 and 0·01 to 1·69 log₁₀ CFU ml^?1. The highest Alicyclobacillus endospore counts in single‐strength juice, concentrate and the final product was at 84 h of processing with 1·32, 1·59 and 1·64 log₁₀ CFU ml^?1, respectively. Conclusions: Alicyclobacillus vegetative cells and endospores accumulate in fruit concentrates during a continuous process running time of 108 h. Significance and Impact of the Study: In conjunction with good manufacturing practices, fruit concentrate manufactures can minimize Alicyclobacillus accumulation in fruit concentrates by limiting the continuous process running time between clean‐ups to under 84 h.