Detection of infectious rotavirus in naturally contaminated source waters for drinking water production

Aims:  To assess chlorine susceptibility of Legionella pneumophila grown from two amoebic hosts, Acanthamoeba castellanii and Hartmannella vermiformis .
Methods and Results:  After being released from amoebae, Leg. pneumophila were chlorinated at 2 and 5 mg l⁻¹ for 5 min–24 h. Bacterial culturability and cytoplasmic membrane deterioration were quantified by culture assay on BCYEα agar and BacLight stains coupled with a fluorescent microscope, respectively. Chlorination reduced the culturability of Leg. pneumophila by 2·93–4·59 log CFU ml⁻¹ and damaged cellular membrane by 53·8–99·2%. Moreover, cells released from H. vermiformis exhibited significantly lower degrees in culturability reduction ( P  = 0·0008) and membrane deterioration ( P  < 0·0001) when compared with those from A. castellanii . The amoebic genus is the most significant parameter affecting cytoplasmic membrane integrity of chlorinated Legionella ( P  < 0·0001), followed by free chlorine concentration ( P  = 0·042).
Conclusions:  Legionella pneumophila replicated from H. vermiformis possess greater chlorine resistance than the cells from A. castellanii .
Significance and Impact of the Study:  This study shows the heterogeneity of amoebae-grown Leg. pneumophila in chlorine susceptibility, which should be considered in the control of legionellae proliferation, particularly in the systems where H. vermiformis is dominant, e.g. hot water plumbing.     

Rapid reduction of Legionella pneumophila on stainless steel with zeolite coatings containing silver and zinc ions

AIMS: To determine the rate of reduction of Legionella pneumophila by stainless steel surfaces with zeolite ceramic coatings containing 2.5% (w/w) silver (Ag) and 14% zinc (Zn) ions. METHODS AND RESULTS: Stainless steel pans with and without Ag/Zn coatings were inoculated with solutions of Leg. pneumophila ATCC 33155 and incubated at 37 degrees C. Survival was monitored using the spread-plate technique on selective buffered charcoal yeast extract agar. Significant reductions of Leg. pneumophila were effected by the Ag/Zn zeolite coatings within 2 h of exposure. CONCLUSIONS, Significance and Impact of the Study: Zeolite ceramic Ag/Zn coatings impart significant anti-Legionella properties to stainless steel surfaces. Coated stainless steel could be used in the manufacture of air ducts, condensation pans and intake and exhaust vents. These products have the potential to reduce numbers of Legionella in air-handling systems.     

Effects of temperature on developmental performance, survival and growth of sea lamprey embryos

This study assesses the influence of thermal regime on the development, survival rates and early growth of embryos of sea lamprey Petromyzon marinus incubated at five constant temperatures (7, 11, 15, 19 and 23° C). The time from fertilization to 50% hatching and from hatching to 50% burrowing were inversely related to incubation temperature. All the embryos incubated at 7° C died at very early stages, while those maintained at 11° C did not attain the burrowing stage. Survival from fertilization to hatching was 61, 89, 91 and 89% at 11, 15, 19 and 23° C, decreasing to 58, 70 and 70% from hatching to burrowing at 15, 19 and 23° C, respectively. Larvae reared during the first 3 months of exogenous feeding in a common environment at constant 21° C, revealed maximum survival for an incubation temperature of 15° C (43% of burrowed larvae) decreasing strongly at 19° C (16%) and 23° C (one suvivor among 240 larvae). Body length at the burrowing stage was maximum for embryos incubated at 19° C, but body mass increased in the interval 15–23° C. Mean incubation temperatures experienced by 117 broods during the embryonic development in the source river were estimated in 15·3±2·30° C and 16·7±1·76° C (mean±1 s.d .) for the periods fertilization-to-hatching and hatching-to burrowing, respectively.     

Ultra-structure and localisation of formazan formed by human neutrophils and amoebae phagocytosing virulent and avirulent Legionella pneumophila

Abstract Legionella pneumophila (LP) strains of differing virulence were incubated with a solution of nitroblue-tetrazolium (NBT) at a concentration of 1 mg · ml⁻ in the presence of Acanthamoeba polyphaga or human polymorphonuclear neutrophils (PMN). Reduction of NBT to formazan occurred at a faster rate in the presence of virulent strains. Reduction appeared to be temperature dependent; at 37°C the reaction rate was higher than at 20°C. On microscopic examination, deposits of formazan around Legionella cells were observed inside amoebae similar to those deposited in human neutrophils. Electron microscopy revealed electron-dense particles surrounding virulent legionellae, which appeared to be associated with formazan foundation. Formazan formation inside amoebae may suggest the presence of a respiratory burst against LP, which is more intense with virulent strains.     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °C).     

Deoxyribonuclease activities in Myxococcus coralloides D

Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37°C, 33°C and 25°C respectively, although high activities were recorded over the temperature range 20–45°C. The pH range of high activity was between 6·0 and 9·0, with an optimum for each DNase at 8·0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg²⁺ and Mn²⁺); however, other metal ions (Fe²⁺, Ni²⁺, Zn²⁺) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P).     

Production and partial characterization of lipases from a newly isolated Penicillium sp. using experimental design

Aims:  The objective of this work was to investigate the lipase production by a newly isolated Penicillium sp . , using experimental design technique, in submerged fermentation using a medium based on peptone, yeast extract, NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained.
Methods and Results:  Lipase activity values of 9·5 U ml⁻¹ in 96 h of fermentation was obtained at the maximized operational conditions of peptone, yeast extract, NaCl and olive oil concentrations (g l⁻¹) of 20·0, 5·0, 5·0 and of 10·0 respectively. The partial characterization of crude enzymatic extract obtained by submerged fermentation showed optimum activity at pH range from 4·9 to 5·5 and temperature from 37°C to 42°C. The crude extract maintained its initial activity at freezing temperatures up to 100 days.
Conclusions:  A newly isolated strain of Penicillium sp . used in this work yielded good lipase activities compared to the literature.
Significance and Impact of the Study:  The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. New lipase producers are relevant to finding enzymes with different catalytic properties of commercial interest could be obtained, without using genetically modified organisms (GMO).     

The effects of temperature on muscle pH, adenylate and phosphogen concentrations in Oreochromis alcalicus grahami, a fish adapted to an alkaline hot-spring

The concentrations of phosphorylcreatine (PCr), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inorganic phosphate (Pi), pyruvate and lactate were determined in freeze-clamped fast muscle samples from Oreochromis alcalicus grahami a fish adapted to extreme alkalinity (∼ pH 10·0) and high temperatures (Lake Magadi, Kenya). Specimens were analysed from both geothermally heated hotsprings (35–37°C) and from isolated cool pools (28°C) and from stocks acclimated to 20°C in the laboratory. The ratios of (ATP)/(ADP) and (ATP)/(ADP) (Pi) decreased with increasing body temperature consistent with an increase in glycolysis and tissue respiration rates, respectively. The apparent equilibrium constant of creatine kinase (K_CK), (creatine) (ATP)/(phosphorylcreatine) (ADP) was found to decrease with increasing temperature: 20·2 (20°C), 13·9 (28°C), 8·0 (37°C). A near constant muscle and blood pH (or slight increase in alkalinity with higher temperatures) was found regardless of body temperature (Blood pH 7·64, 7·74, muscle pH 7·27, 7·51 at 20°C and 35°C, respectively). These results are consistent with an unusual pattern of acid-base regulation in this species.     

The utilisation of arginine by oral streptococci grown glucose-limited in a chemostat

Abstract Legionella pneumophila occurring in drinking water was subjected to environmental stress through holding tests at ambient and elevated temperatures and by chemical disinfection. The bacterium in its native environment was more resistant to adverse conditions, as compared with laboratory-grown organisms. Of the several chemical disinfectants acceptable to drinking water treatment and tested for Legionella inactivation, ozonation was the most efficient method. The C · t ' products indicated that free chlorine was superior to mono- and dichloramines.     

Daily ration and prey size of juvenile piscivore Japanese Spanish mackerel

Daily ration of juvenile Japanese Spanish mackerel Scomberomorus niphonius (32·1–33·1 mm standard length, L _S) was estimated at three temperatures (18·6, 19·5 and 21·2° C) in the laboratory. Gastric evacuation rate ranged between 0·398 (18·6° C) and 0·431 (21·2° C). Diel change in instantaneous consumption rate indicated that juvenile Japanese Spanish mackerel are daylight feeders. The estimated values of the daily ration ranged between 66·1%(18·6° C) and 82·6%(21·2° C) of body mass. These per cent values of daily ration were converted to daily consumption in mg (28 mg at 18·6° C to 34 mg at 21·2° C) using the mean dry body mass of juvenile Japanese Spanish mackerel of 30 mm (42·1 mg). Stomach content analysis of wild specimens collected in the Seto Inland Sea, south‐western Japan, revealed that the majority of wild Japanese Spanish mackerel larvae and juveniles ingested fish larvae with a body size >50% of the predator L _S. Based on the predator‐prey size relationship, the daily consumption of a Japanese Spanish mackerel juvenile of 30 mm was equivalent to 5·1 (18·6° C) to 6·4 (21·2° C) Japanese anchovy Engraulis japonicus larvae which was the dominant prey organism in stomachs of the wild Japanese Spanish mackerel.     

Phenotypic sex differentiation of blue tilapia under constant and fluctuating thermal regimes and its adaptive and evolutionary implications

Oreochromis aureus exposed during the first 28 days of exogenous feeding to constant 35° C, or fluctuating temperatures (day at 35° C, night at 27° C, and vice versa) showed significantly ( P <0·05) faster growth, least size heterogeneity and better survival rates than siblings under constant 27° C. Constant high temperatures had a strong masculinizing effect (M: F sex ratios of 7·33–19·00: 1·00 v . 0·75–0·82: 1·00 in controls reared at 27° C). Fluctuating temperatures had less masculinizing potential but still produced sex ratios significantly skewed to the detriment of females (M: F sex ratios of 2·33–11·50: 1·00). This suggests that ambient temperature may have represented a sufficient environmental pressure for the selection of thermolabile sex-determinism in this species, and presumably in other Oreochromis spp. The evolutionary advantage of thermosensitivity in Oreochromis spp. is discussed, considering a framework where individual advantages oppose, to some degree, to the population or species interest.     

A colony based confirmation assay for Legionella and Legionella pneumophila employing the EnviroAmp™Legionella system and seroagglutination

Modifications to the EnviroAmp^™ Legionella detection system are described which permit the rapid analysis of bacterial colonies taken from Legionella selective media. Capillary PCR permitted twice the number of samples to be analysed with a single kit. When PCR was positive for Leg. pneumophila , this result was confirmed by seroagglutination. The reverse dot blot hybridization assay was only used where PCR indicated a Legionella sp. other than Leg. pneumophila , permitting further savings on detection system components. This technique and standard confirmation procedures were applied to 133 isolates arising from 63 water samples plated to Legionella isolation media. Results agreed except for two isolates which gave a positive result for Legionella spp. by PCR and hybridization but were negative using standard procedures. Raising the annealing/extension temperature of the PCR by 2 °C eliminated the false positive result with these two isolates but did not adversely effect the sensitivity of the assay, as determined by re-testing of 68 environmental isolates and testing of 69 new environmental isolates and 12 Legionella reference species. The modified technique provides a convenient and cost effective alternative to standard confirmation procedures.     

A note on the temperature tolerance of Legionella

A strain of Legionella pneumophila serogroup 1 isolated from the environment had a decimal reduction time in water at 50 degrees C (D50) of 111 min, a D54 of 27 min and a D58 of 6 min. There was little loss of viability at 46 degrees C. Other environmental organisms, a Pseudomonas sp., a Micrococcus sp. and a coliform survived less well at these temperatures. A species of Sarcina had a survival time greater than the L. pneumophila at all the temperatures tested. Other strains of legionellas were tested at 50 degrees C and decimal reduction times calculated. These ranged from 80 min for another strain of L. pneumophia serogroup 1 to 216 min for L. bozemannii . Legionella micdadei did not survive well at 50 degrees C.     

Early development and growth of the laboratory reared north-east Atlantic mackerel Scomber scombrus L.

The early development, growth and morphological changes of mackerel Scomber scombrus were investigated at different incubation temperatures (8, 10, 13, 15 and 18° C). Details on the early life history are illustrated with special reference to morphological transformations. Culture techniques to rear larval mackerel stages are described using laboratory cultured foods. Artificially fertilized eggs were hatched after 80·6 h at 18·4° C and 256·8 h at 8·7° C. The standard length ( L _S) of the individuals at first feeding was 4·71 ± 0·18 mm. Four mortality critical periods and cannibalistic behaviour were identified. A maximum average larval size of 37·5 ± 4·41 mm L _S was attained 30 days post-hatch (dph) at 18·4° C. Development and growth were affected significantly by temperature during both endogenous and exogenous feeding periods. Larvae grew more rapidly at high, than at low temperatures. Daily specific growth rate (in mass) ranged from 2·4% at 10·6° C to 16·9% at 18·4° C. Likewise, average growth rate (in length) ranged from 0·05 mm day⁻¹ at 8·4° C to 0·37 mm day⁻¹ at 18·4° C. The allometric relationship of L _S, with several body measurements was not affected by temperature. Comparison with larvae collected in the Bay of Biscay did not show any significant difference in the dry mass and L _S relationship; conversely, the growth rate in length differed significantly between both laboratory and field conditions. The trends observed in the laboratory are described in relation to some aspects of the year-class strength regulation.     

Growth, diet and metabolism of common wolf–fish in the North Sea, a fast–growing population

The von Bertalanffy growth parameters for common wolf–fish Anarhichas lupus in the North Sea were: male: L ∞=111·2 cm, t ₀=–0·43 and K =0·12; and female: L ∞=115·1 cm, t ₀=–0·39 and K =0·11, making this the fastest growing stock reported. Resting metabolic rates (RMR±S.E.) and maximum metabolic rates (MMR±S.E.) for six adult common wolf–fish (mean weight, 1·39 kg) at 5° C were 12·18±1·6 mg O₂ kg^–1 h^–1 and 70·65±7·63 mg O₂ kg^–1 h^–1 respectively, and at 10° C were 25·43±1·31 mg O₂ kg^–1 h^–1 and 113·84±16·26 mg O₂ kg^–1 h^–1. Absolute metabolic scope was 53% greater at 10° C than at 5° C. The diet was dominated by Decapoda (39% overall by relative occurrence), Bivalvia (20%) and Gastropoda (12%). Sea urchins, typically of low energy value, occupied only 7% of the diet. The fast growth probably resulted from summer temperatures approximating to the optimum for food processing and growth, but may have been influenced by diet, and reduced competition following high fishing intensity.     

Thermal tolerance of a northern population of striped bass Morone saxatilis

Thermal tolerance of age 0+ year Shubenacadie River (Nova Scotia, Canada) striped bass Morone saxatilis juveniles (mean ± s . e . fork length, L _F, 19·2 ± 0·2 cm) acclimated in fresh water to six temperatures from 5 to 30° C was measured by both the incipient lethal technique (72 h assay), and the critical thermal method ( C _m). The lower incipient lethal temperature ranged from 2·4 to 11·3° C, and the upper incipient lethal temperature ( I _U) from 24·4 to 33·9° C. The area of thermal tolerance was 618° C². In a separate experiment, the I _U of large age 2+ year fish (34·4 ± 0·5 cm L _F) was 1·2 and 0·6° C lower ( P < 0·01) than smaller age 1+ year fish (21·8 ± 0·5 cm L _F) at acclimation temperatures of 16 and 23° C. Using the C _m, loss of equilibrium occurred at 27·4–37·7° C, loss of righting response at 28·1–38·4° C and onset of spasms at 28·5–38·8° C, depending on acclimation temperature. The linear regression slopes for these three responses were statistically similar (0·41; P > 0·05), but the intercepts differed (25·3, 26·0 and 26·5° C; P < 0·01). The thermal tolerance of this northern population appears to be broader than southern populations.     

Rate of energy depletion and overwintering mortality of juvenile walleye pollock in cold water

The winter energy deficit and mortality of juvenile walleye pollock at extremely cold temperature were examined by field observations and laboratory experiments. In the Doto area, along the northern coast of Japan, juvenile walleye pollock resided on the continental shelf despite extremely cold temperatures (mean 0·4° C) during the latter half of winter (March to April). Measurements of the rate of energy depletion (equivalent to the routine metabolic rate) revealed that juvenile walleye pollock consumed 37% less energy at 0·5° C than at 2·0° C, suggesting an energetic benefit of residence in cold water (<1·0° C) over the shelf during winter. Prior to the starvation experiments, temperatures and ration level in the holding tanks were adjusted to create two different body condition groups of fish. Under the thermal condition of the latter half of winter (0·5° C), fish with a mean condition factor of 0·6 and 0·5 suffered 19·1 and 74·5% mortality, respectively, at the end of the experiments (after 56 days). The residual analysis of total body energy demonstrated that the cause of mortality was mainly associated with the depletion of energy reserves. When a logistic regression model for mortality derived from the experiments was applied to wild fish collected in March, the estimated overwintering mortality in 2004 and 2005 was 25·4 and <2·3%, respectively, assuming no feeding during the winter. Considering that juvenile walleye pollock feed during winter as shown in previous studies, intense overwintering mortality induced by energy depletion is improbable during the latter half of winter in the Doto area.     

Factors affecting the survival of Salmonella and Escherichia coli in anaerobically fermented pig waste

The survival of Salmonella typhimurium was investigated in acidogenic, anaerobically fermented pig wastes and in synthetic media, each containing volatile fatty acids (VFA). Salm. typhimurium survived at pH 6·8, but not at pH 4·0, when incubated at 37°C for 24 h in either fermented or synthetic medium containing VFA. The minimum inhibiting concentration of VFA for Salm. typhimurium after 48 h incubation at 30°C at pH 4·0 was 0·03 mol/l and for Escherichia coli it was 0·09 mol/l. Fermented pig wastes in a digester, maintained at pH 5·9, were inoculated with Salm. typhimurium and then incubated at 37°C for 24 h. The pH was adjusted to either 4·0 or 5·0 and after a further 48 h at 30°C, Salm. typhimurium survived at pH 5·0 but not at pH 4·0. It was concluded that pH is critical in determining the survival of this organism in acidogenic anaerobically fermented pig waste.     

Efficacy of high sodium chloride concentrations for the destruction of Listeria monocytogenes

The effect of sodium chloride concentrations (6, 16 and 26% (w/v) NaCl) on the survival of Listeria monocytogenes at low temperatures (10°C and under refrigeration, average 2°C) and frozen (- 18°C) was investigated. All salt concentrations tested were ineffective in reducing numbers over 6 h incubation at low temperatures. Over a longer time (33 d) at low temperatures, the organism grew in 6% NaCl, numbers remained the same in 16% NaCl and numbers declined in 26% NaCl. Although L. monocytogenes was destroyed in 26% NaCl, numbers declined too slowly for immersion in cold brine at this concentration to be a useful bacteriocidal treatment. Storage at - 18°C for 33 d caused no significant reduction in numbers at any of the NaCl concentrations tested.     

Influence of rearing temperature during the larval and nursery periods on growth and sex differentiation in two Mediterranean strains of Dicentrarchus labrax

European sea bass Dicentrarchus labrax of the north‐western (NW) and south‐eastern (SE) Mediterranean Sea strains were exposed to different temperatures (13, 17 or 21° C) during the larval rearing (11–51 days post hatching, dph) or nursery periods (55–95 dph), in order to examine the effects of temperature on sex differentiation and subsequent growth during the first year of life. Higher growth was observed during exposure to higher temperatures, but fish of the NW strain exposed to 13 or 17° C during larval rearing exhibited compensatory growth once exposure to the lower temperatures finished, and as a result their final size at 300 dph was similar or greater to the group exposed to 21° C. Fish exposed to 17° C during the nursery period also had similar size to fish exposed to 21° C after 300 days of rearing, but the fish exposed to 13° C remained significantly smaller (ANOVA, n  = 55–100, P  < 0·05). There were significant differences in the sex ratio among the fish exposed to different temperatures during the two periods of rearing, with high temperature (21° C) resulting in a significantly higher percentage of males in the population, both in the NW (ANOVA, n  = 2, P  < 0·04) and SE populations (ANOVA, n  = 2, P  < 0·01). The masculinization effect of high temperature was significantly stronger during the larval rearing stage, both in the NW (ANOVA, n  = 2, P  < 0·005) and SE populations (ANOVA, n  = 2, P  < 0·01). None of the temperature manipulations could produce 100% females, suggesting that there is a part of the genetic component in sex differentiation which is not labile to environmental influence.