Detection and enumeration of Dekkera anomala in beer, cola, and cider using real-time PCR

Aims: In this article, a quantitative real‐time PCR assay for detection and enumeration of the spoilage yeast Dekkera anomala in beer, cola, apple cider, and brewing wort is presented as an improvement upon existing detection methods, which are very time‐consuming and not always accurate. Methods and Results: Primers were designed to exclude other organisms common in these beverages, and the assay was linear over 6 log units of cell concentrations. The addition of large amounts of non‐target yeast DNA did not affect the efficiency of this assay. A standard curve of known DNA was established by plotting the C_t values obtained from the QPCR against the log of plate counts on yeast peptone dextrose medium and unknowns showed exceptional correlation when tested against this standard curve. The assay was found to detect D. anomala at levels of 10–14 CFU ml^?1 in either cola or beer and at levels of 9·4–25·0 CFU ml^?1 in apple cider. The assay was also used to follow the growth of D. anomala in brewing wort. Conclusions: The results indicate that real‐time PCR is an effective tool for rapid, accurate detection and quantitation of D. anomala in beer, cola and apple cider. Significance and Impact of the Study: This method gives a faster and more efficient technique to screen beer, cola, and cider samples and reduce spoilage by D. anomala. Faster screening may allow for significant reduction in economic loss because of reduced spoilage.     

Characterization and validation of a chemiluminescent assay based on a 1,2-dioxetane for rapid detection of enterococci in contaminated water and comparison with standard methods and qPCR

Aims: To evaluate the potential for using a novel chemiluminescence‐based enzyme assay for rapid detection of enterococci in water contaminated with faecal waste. Methods and Results: The novel assay (EntLight) was based on the enzymatic hydrolysis of the chemiluminescent 1,2‐dioxetane [(4‐methoxy‐4(3‐β‐d ‐glucoside‐4‐chlorophenyl)]spiro[1,2‐dioxetane‐3‐1,3‐tricyclo[7·3·1·0^2,7]tridec‐2,7‐ene] specific for β‐d ‐glucosidase. The specificity of the proposed EntLight assay was characterized using 26 different Enterococcus strains and 10 bacterial genera other than Enterococcus. With an analysis time of ≤8 h, the assay was found to be sensitive and specific. Validation experiments were carried out using water samples contaminated with raw municipal wastewater in comparison with qPCR and ISO standard methods. EntLight was successfully applied to detect enterococci in contaminated water within ≤8 h, and the proposed assay correlated well with both qPCR and ISO standard methods (R² > 0·776). Conclusions: EntLight can be applied to rapid and simple detection of viable enterococci in water contaminated with faecal matter. Significance and Impact of the Study: The novel EntLight assay and qPCR have the potential to be used as methods for early warning (1–7 h) of faecal pollutions in different water types.     

Rapid detection of Vibrio parahaemolyticus by PCR targeted to the histone-like nucleoid structure (H-NS) gene and its genetic characterization

Aims: The aim of this study was to explore a new PCR target gene for Vibrio parahaemolyticus, based on the histone‐like nucleoid structure (H‐NS) gene. Methods and Results: Primers for the H‐NS gene were designed for specificity to V. parahaemolyticus and incorporated into a PCR assay. The PCR assay was able to specifically detect all of the 82 V. parahaemolyticus strains tested, but did not result in amplification in the 47 other Vibrio spp. and nonVibrio spp. strains. The detection limit of the PCR assay was 0·14 pg purified genomic DNA and 1·8 × 10⁵ CFU g^?1 spiked oyster samples from V. parahaemolyticus RIMD2210633. Furthermore, a multiplex PCR assay targeting the hns, tdh and trh genes was successfully developed to detect virulent V. parahaemolyticus strains. Conclusions: The H‐NS‐based PCR assay developed in this study was sensitive and specific, with great potential for field detection of V. parahaemolyticus in seawater or seafood samples. Significance and Impact of the Study: The H‐NS gene was validated as a new specific marker gene in PCR assays for accurate detection and identification of V. parahaemolyticus, which has the potential to be applied in diagnostics and taxonomic studies.     

Development of a microtitre plate indirect ELISA for measuring cortisol in teleosts, and evaluation of stress responses in rainbow trout and gilthead sea bream

A microtitre plate indirect enzyme‐linked immunoassay (ELISA) was developed for measuring plasma cortisol levels in rainbow trout Oncorhynchus mykiss, gilthead sea bream Sparus auratus sea bass Dicentrarchus labrax and Senegalese sole Solea senegalensis. Covalink microplates pretreated with disuccinimidyl suberate were coated with bovine serum albumin (BSA) conjugated to cortisol‐3‐carboxymethyl oxime. After blocking with BSA, competition was started by addition of plasma samples and anti‐cortisol antibody raised in rabbit. Goat anti‐rabbit IgG conjugated‐peroxidase was added as second antibody and then incubated with orthophenylenediamine as substrate. Reaction was stopped with 0·1 M HCl and absorbance was read at 450 nm in an automatic plate reader. The standard curve was linear from the lower limit of sensitivity of the assay (c. 0·3 ng ml^?1) to c. 3000 ng ml^?1. Dose‐response inhibition curves using serially diluted plasma samples of four species consistently showed parallelism with the standard curve using cortisol. The ELISA satisfied the strictest criteria of specificity (cross‐reactivity of anti‐cortisol antibody with testosterone, progesterone and 17ß‐oestradiol was negligible, cross‐reactivity with cortisone, corticosterone and 11‐deoxycortisol, was 1·5, 1 and 0·1%, respectively), reproducibility (interassay CV <6%), precision (intra‐assay CV <4%), and accuracy (average recovery >98%). Plasma cortisol concentration in rested fishes was in the range of 5–30 ng ml^?1. To physiologically validate the technique, changes in plasma cortisol concentrations were also measured in plasma of rainbow trout and gilthead sea bream following an acute 15 min chasing or 3 min air‐exposure stress, respectively. In both species plasma concentrations of cortisol, glucose and lactate rose significantly with respect to controls, showing concentrations similar to those reported previously for these species under similar stress conditions. Furthermore, gilthead sea bream chronically stressed by maintaining for 14 days under increased stocking density conditions also showed increased concentrations of plasma cortisol and glucose. These results validate the indirect ELISA technique developed for use in the evaluation of plasma cortisol concentration of at least four fish species.     

Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57-based qPCR assays

Aims: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. Methods and Results: TaqMan^® assays were designed to target the IS900 and f57 genetic elements of Map. Both real‐time PCR assays were integrated with the Adiapure^® Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml^?1, 2·8 CFU g^?1 and 30 CFU g^?1 for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD’s for the f57 assay were 6·2 CFU ml^?1, 26·7 CFU g^?1 and 316 CFU g^?1. Conclusion: The integrated Adiapure^® extraction – IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map‐DNA in cheese and whole milk powder. Significance and Impact of the Study: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.     

Study of laccase production by Pleurotus ostreatus in a 5 l bioreactor and application of the enzyme to determine the antioxidant concentration of human plasma

Aims: To achieve high laccase production from Pleurotus ostreatus in a bench top bioreactor and to utilize the enzyme for determination of the total antioxidant concentration (TAC) of human plasma. Methods and Results: Laccase production by P. ostreatus studied in a benchtop bioreactor was as high as, 874·0 U ml^?1 in presence of copper sulfate. The enzyme was used to replace metmyoglobin and hydrogen peroxide for the estimation of TAC in human plasma. The trolox equivalent antioxidant concentrations determined by the laccase‐based method and metmyoglobin method ranged from 1·63 ± 0·011 to 1·80 ± 0·006 mmol l^?1 and from 1·41 ± 0·004 to 1·51 ± 0·008 mmol l^?1 plasma, respectively. Conclusions: Pleurotus ostreatus produced high amount of extracellular laccase in a benchtop bioreactor. The enzyme can be used to assay TAC of blood plasma without the interference encountered with the hydrogen peroxide and metmyoglobin mediated assay method. Significance and Impact of the Study: Laccase production by P. ostreatus obtained in this study was the highest among all reported laccase producing white‐rot fungi. Moreover, an accurate laccase‐based assay method was developed for detection of TAC in human plasma.     

Evaluation of real‐time PCR methods for quantification of Acanthamoeba in anthropogenic water and biofilms

Aims: To assess two real‐time PCR methods (the Riviere and Qvarnstrom assays) for environmental Acanthamoeba. Methods and Results: DNA extracted from Acanthamoeba castellanii taken from water and biofilms of cooling towers was analysed by the Riviere and Qvarnstrom assays. To quantify environmental Acanthamoeba, the calibration curves (DNA quantity vs cell number) were constructed with samples spiked with A. castellanii. The calibration curves for both quantitative PCR assays showed low variation (coefficient of variation of C_t≤ 5·7%) and high linearity (R² ≥ 0·99) over six orders of magnitudes with detection limit of three cells per water sample. DNA quantity determined by Qvarnstrom assay was equivalent between trophozoites and cysts (P = 0·49), whereas a significant difference was observed with Riviere assay (P < 0·0001). Riviere assay failed to detect Acanthamoeba in 21% (15/71) of the environmental samples which were positively detected by Qvarnstrom assay, while one sample (1·4%) was shown positive by Riviere assay but negative by Qvarnstrom assay. Moreover, Acanthamoeba counts by Qvarnstrom assay were greater than those by Riviere assay (P < 0·0001). Conclusions: Qvarnstrom assay performs better than Riviere assay for detection and quantification of Acanthamoeba in anthropogenic water and biofilms. Significance and Impact of the Study: Qvarnstrom assay may significantly contribute to a better knowledge about the distribution and abundance of Acanthamoeba in environments.     

Classification of Salmonella enterica serotypes from Australian poultry using repetitive sequence-based PCR

Aims: To evaluate a semi‐automated repetitive extragenic palindromic sequence‐based PCR (rep‐PCR) system for the classification of Salmonella serotypes from Australian poultry. Methods and Results: Using a DNA fingerprint library within the DiversiLab^® System, four separate databases were constructed (serogroup B, C, E and Other). These databases contained 483 serologically confirmed (reference laboratory) Salmonella isolates. A blinded set of Salmonella cultures (n = 155) were typed by rep‐PCR, matched against the internal library and compared with traditional serotyping. The predicted (Kullback–Leibler) serotype of 143 (92·3%) isolates matched traditional typing (P < 0·05). Of the 12 (7·7%) remaining isolates, ten (6·5%) resulted in ‘No Match’, one (0·65%) was incorrectly matched to the library (Salm. subsp 1 ser 4,12:‐:‐), and the other (0·65%) was referenced as Salm. ser. Sofia, whereas rep‐PCR and in‐house serotyping concurred as Salmonella serovar Typhimurium. Financial analysis showed higher material cost (215%) and a lower labour component (47·5%) for rep‐PCR compared with serotyping. Conclusion: The DiversiLab^® System, with serogroup databases, was successfully implemented as an adjunct for reference serotyping of Salmonella enterica. Significance and Impact of the Study: The DiversiLab^® System platform is a cost‐effective and easy‐to‐use system, which can putatively determine Salmonella enterica serotypes within a few hours.     

A real-time qPCR assay to quantify Fusarium graminearum biomass in wheat kernels

Aims: To develop a real‐time PCR assay to quantify Fusarium graminearum biomass in blighted wheat kernels. Methods and Results: Primers designed to amplify a gene in the trichothecene biosynthetic cluster (TRI6) were evaluated for sensitivity and specificity. Primer pair Tri6_10F/Tri6_4R specifically and consistently amplified a 245‐bp DNA fragment from F. graminearum. A workflow was developed and validated to extract DNA from infested grain. The assay detected as little as 10 μg of F. graminearum mycelia in 1 g of ground wheat grain with a high correlation between fungal biomass and cycle threshold values (R² = 0·9912; P = 0·004). In field‐inoculated grain, qPCR measurements of biomass correlated closely with deoxynivalenol levels (R = 0·82, P < 0·0001) and two visual techniques to assess grain quality (R = 0·88, P < 0·0001 and R = 0·81, P < 0·0001). Conclusions: The qPCR assay provided accurate and precise assessments of the amount of F. graminearum biomass in blighted wheat kernels. This method represents a technical advance over other approaches to quantify kernel colonization and real‐time PCR detection methodologies for F. graminearum that do not correlate quantification of fungal genomic DNA to biomass. Significance and Impact of the Study: Quantifying F. graminearum biomass, especially low levels of growth associated with kernels that are visually asymptomatic, represents a new approach to screen for resistance to kernel infection, an understudied yet potentially important avenue to reduce the impact of head blight.     

Discrimination of gamma-irradiated and nonirradiated Vibrio vulnificus by using real-time polymerase chain reaction

Aims: To develop a PCR strategy for Vibrio vulnificus in irradiated foods. Methods and Results: Real‐time PCR was used to discriminate between DNA from γ‐irradiated and nonirradiated cells. γ‐Irradiation at 1·08 KGy and above of cell suspensions containing 1 × 10⁶ CFU ml^?1 resulted in 0 CFU ml^?1. Real‐time PCR was able to detect 86·6% destruction by 1·08 KGy, while ethidium bromide monoazide (EMA) real‐time PCR was able to detect 93·2% destruction at this dose. With 3·0 and 5·0 KGy, EMA real‐time PCR was able to detect 99·3% and 100% destruction, respectively. Conclusions: The inability to detect via PCR extensively degraded DNA resulting from γ‐irradiation can be taken as evidence of cell death. The increased ability of EMA to further reduce the detectable number of target sequences via PCR, with DNA from cells exposed to increased doses of γ‐irradiation, can be considered to reflect the accompanying increase in membrane damage which allows EMA to penetrate the cells. Significance and impact of the study: This is the first study that has made use of the PCR to discriminate between γ‐irradiated and nonirradiated bacterial cells and has led to insights regarding the ability of the PCR to discriminate between nonirradiated and γ‐irradiation destroyed cells.     

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis

Aim: To develop a TaqMan probe‐based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. Methods and Results: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay’s specificity, detection limit, intra‐ and inter‐assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100‐fold more sensitive than the cPCR. No cross‐reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10¹¹ and 2·75 × 10⁸ target copies ml^?1 of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. Conclusion: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. Significance and Impact of the Study: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.     

Consistency in the host specificity and host sensitivity of the Bacteroides HF183 marker for sewage pollution tracking

Aims: The host specificity (H‐SPF) and host sensitivity (H‐SNV) values of the sewage‐associated HF183 Bacteroides marker in the current study were compared with the previously published studies in South East Queensland (SEQ), Australia, by testing a large number of wastewater and faecal DNA samples (n = 293) from 11 target and nontarget host groups. This was carried out to obtain information on the consistency in the H‐SPF and H‐SNV values of the HF183 marker for sewage pollution tracking in SEQ. Methods and Results: Polymerase chain reaction (PCR) analysis was used to determine the presence/absence of the HF183 marker in wastewater and faecal DNA samples. Among the human composite wastewater (n = 59) from sewage treatment plants and individual human (n = 20) faecal DNA samples tested, 75 (95%) were PCR positive for the HF183 marker. The overall H‐SNV of this marker in target host group was 0·95 (maximum of 1·00). Among the 214 nontarget animal faecal DNA samples tested, 201 (94%) samples were negative for the HF183 marker. Six chicken, five dog and two bird faecal DNA samples, however, were positive for the marker. The overall H‐SPF of the HF183 marker to differentiate between target and nontarget faecal DNA samples was 0·94 (maximum of 1·00). Conclusions: The H‐SNV (0·95) and H‐SPF (0·94) values obtained in this study was slightly lower than previous studies (H‐SNV value of 1·00 in 2007 and 1·00 in 2009; H‐SPF value of 1·00 in 2007 and 0·99 in 2009). Nonetheless, the overall high H‐SNV (0·98) and H‐SPF (0·97) values of the HF183 marker over the past 4 years (i.e. 2007–2011) suggest that the HF183 marker can be reliably used for the detection of sewage pollution in environmental waters in SEQ. Significance and Impact of the Study: In the current study, the HF183 marker was detected in small number nontarget animal faecal samples. Care should be taken to interpret results obtained from catchments or waterways that might be potentially contaminated with dog faecal matter or poultry litter.     

Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real‐time quantitative PCR

Aims: To optimize ethidium monoazide (EMA) coupled with real‐time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems. Methods and Results: EMA (0·9–45·5 μg ml^?1) and propidium monoazide (PMA, 0·9 and 2·3 μg ml^?1) combined with qPCR (i.e. EMA‐qPCR and PMA‐qPCR, respectively) were applied to unheated and heated (70°C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight‐EM). The effects of nontarget microflora and sample matrix on the performance of EMA‐qPCR were also evaluated. In comparison with BacLight‐EM results, qPCR with EMA at 2·3 μg ml^?1 was determined as the optimal EMA‐qPCR assay, which performed equally well as PMA‐qPCR for unheated Leg. pneumophila but better than PMA‐qPCR for heated Leg. pneumophila (P < 0·05). Moreover, qPCR with EMA at 2·3 μg ml^?1 accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella‐like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0·05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA‐qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR. Conclusions: The qPCR with EMA at 2·3 μg ml^?1 may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems. Significance and Impact of the Study: The EMA‐qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.     

Rapid detection of “Candidatus Phytoplasma mali” by recombinase polymerase amplification assays

Isothermal recombinase polymerase amplification (RPA) assays for the specific detection of “Candidatus Phytoplasma mali (Ca. P. mali),” the causal agent of apple proliferation, were developed. The assays amplify a fragment of the imp gene and amplimers were detected either by fluorescence in real‐time mode (TwistAmp^®exo assay) using a fluorophore‐labelled probe or by direct visualization employing a lateral flow device (TwistAmp^®nfo assay/Milenia^®HybriDetect). The RPA assays specifically amplified DNA from “Ca. P. mali” strains, and cross‐reactivity with other phytoplasmas or plant DNA was not observed. The limit of detection was determined with a cloned imp standard, and positive results were obtained down to 10 copies with both RPA assay formats. In comparison with a TaqMan real‐time PCR assay based on the same target gene, the RPA assays were equally sensitive, but results were obtained faster. Simplified nucleic acid extraction procedures from plant tissue with Tris‐ and CTAB‐based buffers revealed that crude Tris–DNA extracts were a suitable source for RPA tests while larger concentrations of CTAB were inhibitory. This is the first report of RPA‐based assays for the detection of “Ca. P. mali”. The assays are suitable for high‐throughput screening of plant material and point‐of‐care diagnostic and can be potentially combined with a simplified DNA extraction procedure.     

Using stable isotope ratios as a tracer of feeding adaptation in released Japanese flounder Paralichthys olivaceus

Release‐recapture experiments were conducted to examine temporal changes of the carbon and nitrogen stable isotope (δ¹³C and δ¹⁵N) ratios in the muscle tissue of artificially produced Japanese flounder Paralichthys olivaceus, juveniles. About 9000 juveniles (mean ± s .d . 43·3 ± 5·2 mm in standard length and 1·07 ± 0·37 g, n = 15) were released in each of three coastal areas: Chojagasaki, Arasaki and Jogashima with different geographical conditions, along Sagami Bay, Pacific coast of central Japan. Recapture efforts were made on 4, 11, 18, 40 and 55 days after the release. The stable isotope ratios, RNA:DNA ratio, stomach content mass (per body mass M_sc) and condition factor (K) of recaptured individuals were measured. The mean ± s .d . δ¹³C and δ¹⁵N values (n = 15) were ?18·3 ± 0·2‰ and 12·2 ± 0·2‰, respectively at the release. Wild Japanese flounder juveniles were captured only in Chojagasaki, and the δ¹³C and δ¹⁵N values (n = 6) were ?14·0 ± 0·4‰ and 13·2 ± 0·7‰, respectively; these values were considered to represent the wild diet. Nutritional conditions of the released and recaptured juveniles as determined by the RNA : DNA ratio, M_SC and K were indicated to be the best in Chojagasaki, in which the stable isotope ratios gradually shifted towards and reached the wild values within 40 days. This result along with stomach content analyses suggested that the released juveniles had acquired a wild feeding habit. In Arasaki and Jogashima, nutritional conditions of the recaptured juveniles were poorer, with no clear changes in the stable isotope ratios. Greatly varied stable isotope ratio values were observed in the juveniles recaptured in Chojagasaki 11 days after the release, ranging from the release levels to the wild levels. The extent of changes in the stable isotope ratios had a positive correlation to the RNA : DNA ratio and K of these juveniles (r = 0·87, n = 10 and r = 0·83, n = 18, respectively). The analyses of stable isotope ratios coupled with nutritional condition were considered to be an effective tool to examine post‐release feeding adaptation of Japanese flounder juveniles.     

Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology

The objective of this study was to determine the effect of freezing on the function in Atlantic salmon Salmo salar spermatozoa. The semen was frozen in Cortland's medium + 1.3M dimethyl sulphoxide + 0.3M glucose + 2% bovine serum albumin (final concentration) in a ratio of 1:3 (semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 0·5 ml of sperm suspension were frozen in 4 cm of N₂L. They were thawed in a thermoregulated bath (40° C). After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine triphosphate) nick‐end labelling (TUNEL)], plasma membrane integrity (SYBR‐14/PI) and mitochondrial membrane potential (ΔΨMMit, JC‐1) were evaluated by flow cytometry and motility was evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and treatment semen were tested at a sperm density of 1·5 × 10⁷ spermatozoa oocyte^?1, by observation of the first cleavages after 16 h incubation at 10° C. In the cryopreserved semen (T), the mean ± s.d . DNA fragmentation was 4·8 ± 2·5%; plasma membrane integrity 75·2 ± 6·3%; mitochondrial membrane potential 51·7 ± 3·6%; motility 58·5 ± 5·3%; curved line velocity (V_CL) 61·2 ± 17·4 µm s^?1; average‐path velocity (V_AP) 50·1 ± 17·3 µm s^?1; straight‐line velocity (V_SL) 59·1 ± 18·4 µm s^?1; fertilization rate 81·6 ± 1·9%. There were significant differences in the plasma membrane integrity, mitochondrial membrane potential, motility, fertilization rate, V_CL, V_AP and V_SL compared with the controls (P < 0·05). Also the mitochondrial membrane potential correlated with motility, fertilization rate, V_CL and V_SL (r = 0·75; r = 0·59; r = 0·77 and r = 0·79, respectively; P < 0·05); and the fertilization rate correlated with V_CL and V_SL (r = 0·59 and r = 0·55, respectively).     

Standard metabolism and growth dynamics of laboratory‐reared larvae of Sardina pilchardus

This study provides the first measurements of the standard respiration rate (R_S) and growth dynamics of European sardine Sardina pilchardus larvae reared in the laboratory. At 15° C, the relationship between R_S (µl O₂ individual^?1 h^?1) and larval dry mass (M_D, µg) was equal to: R_S = 0·0057(±0·0007, ± s.e.)·M_D^{0·8835(±0·0268)}, (8–11% M_D day^?1). Interindividual differences in R_S were not related to interindividual differences in growth rate or somatic (Fulton's condition factor) or biochemical‐based condition (RNA:DNA).     

Allometric relationship between body mass and aerobic metabolism in zebrafish Danio rerio

The relationship between body mass (M) and metabolic rate was investigated through the assessment of active (R_A) and standard (R_S) metabolic rate at different life stages in zebrafish Danio rerio (5 day‐old larvae, 2 month‐old juveniles and 6 month‐old adults). Scaling exponents and constants were assessed for standard (R_S = 0·273M^0·965 in mgO₂ g^?1 h^?1) and active metabolic rate (R_A = 0·799M^0·926 in mgO₂ g^?1 h^?1). These data provide the basis for further experiments regarding the effects of environmental factors on aerobic metabolism throughout the life cycle of this species.     

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

Aims: To develop a quick and accurate PCR‐based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. Methods and Results: The number of BbrY in faeces was detected by using strain‐specific quantitative real‐time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA‐intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10⁵–10⁹ cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) log CFU g^?1 [mean (±SD)] of BbrY was detected in faeces by using strain‐specific transgalactosylated oligosaccharide–carbenicillin (T‐CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) log cells per g. Conclusions: Strain‐specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Significance and Impact of the Study: Combination of strain‐specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.