Antibacterial activity of the lactoperoxidase system against Aeromonas hydrophila in broth, skim milk and ewes' milk

The effect of the lactoperoxidase (LP)-system on Aeromonas hydrophila in broth, skim milk and ewes' milk was investigated. The behaviour of three strains in activated and control substrates was studied at 6 and 28°C. The activated ovine lactoperoxidase was bactericidal for Aer. hydrophila . In the three LP-treated substrates the tested strains were completely inactivated after 2 (28°C) and 24 h (6°C) and then remained undetectable during storage for up to 25 d. The use of the LP-system as a preservative against Aer. hydrophila could be very useful even when adequate cooling facilities are available.     

Influence of growth temperature on the production of extracellular virulence factors and pathogenicity of environmental and human strains of Aeromonas hydrophila

The biochemical properties, virulence for mice and trout, and the extracellular virulence factors at 28° and 37°C of 11 environmental and nine human strains of Aeromonas hydrophila were compared. All the environmental isolates and four of the human group were virulent for trout at 3 x 10⁷ cfu, but only human strains were able to cause death or lesions in mice by the intramuscular route. Extracellular virulence factors such as haemolysins, cytotoxins and proteases were also investigated in supernatant fluids of cultures grown at 28°C and 37°C. The production of haemolysins, caseinases, elastases and growth yields of environmental strains decreased sharply during cultivation at 37°C but cytotoxins were produced to the same extent, or slightly less, than at 28°C. The human strains differed from the environmental strains in response to growth temperatures: protease activity decreased at 37°C, although growth yield was not affected, but more haemolysins and cytotoxins were produced by the virulent strains at this temperature than at 28°C. Sodium caseinate SDS-PAGE of culture supernatant fluids of selected human strains revealed that temperature selectively inhibited the production of certain proteases.     

The effect of temperature on Aeromonas hydrophila infection in goldfish, Carassius auratus

The effect of temperature on Aeromonas hydrophila infection in goldfish, Carassius auratus , was studied using A. hydrophila strain A-3500. After comparison of four different infection methods, subcutaneous injection was selected. Different test temperatures were also tested and higher mortality was observed at 17 and 25°C during a 15-day period. SDS-PAGE analysis of outer membrane proteins prepared from A. hydrophila cultured at 10, 17, 25 and 32°C in formulated salt water showed different protein profiles. For example, a 40-kDa band was found only at 17 and 25°C. Phagocytic rates of A. hydrophila by goldfish macrophages at 10, 17, 25 and 32°C were 20.46 ± 2.07, 16.15 ± 1.39, 15.94 ± 1.85 and 22.22 ± 2.49%, respectively. The results indicated that temperature affects both the cell membrane structure of A. hydrophila and phagocytic activity of goldfish macrophages, resulting in varying fish mortality when infected at different temperatures.     

Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapianilotica

Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic andproteolytic activity were studied with respect to temperature and time of incubation as well as thelethal toxicity on tilapia, Tilapia nilotica . The highest production of the haemolysin productwas achieved when Aer. hydrophila was grown at 35°C for 30 h. Tilapia erythrocytewas found to be more susceptible than sheep erythrocyte for determining the haemolytic activity.The haemolytic activity against tilapia erythrocyte was completely inactivated after heating theECP at 60°C for 10 min or 55°C for 15 min. The proteolytic activity was maximized whenthe bacterium was grown at 30°C for 36 h. Complete inactivation of the protease enzyme wasperformed after heating the ECP at 80°C for 10 min or 70°C for 15 min. Aeromonashydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD₅₀ 2·1 × 10⁴ cell/fish), as well as heat stable unknown virulent factors thatwere responsible for 20% mortality. The lethality of ECP was decreased by heating andcompletely inactivated by boiling at 100°C for 10 min.     

Effect of Some Environmental Factors on Psychrophilic Microbacteria

S ummary . The growth of three psychrophilic strains of Microbacterium isolated from meat was studied at 10 temperatures between 0° and 35° and at 6 water activity (a_w) levels between 0.99 and 0.94. The temperature and water relations of the three strains were similar. For all strains the rate of growth was fastest at 25° and a_w 0.99, and growth did not occur at 35°.
Further experiments on one strain showed that under aerobic conditions the range of water activities permitting growth was independent of temperature, but under anaerobic conditions the minimum water activity increased from about 0.94—0.96 as the temperature was reduced from 25° to 0°. Growth was inhibited by low concentrations of undissociated nitrous acid, and inhibition was greater at 0° than at 10° or 25°.     

The effect of incubation temperature, sodium chloride and ascorbic acid on the growth kinetics of Aeromonas hydrophila

The effects of temperature, sodium chloride and ascorbic acid on the aerobic growth kinetics of a clinical strain of Aeromonas hydrophila were evaluated. At 5°C, ascorbic acid (1 mmol l^-1) and sodium chloride (3% w/v) inhibited the growth of the organism. At 10°C, ascorbic acid depressed only the maximum population densities (A) by approximately 2 log cycles, but not maximum specific growth rate (μ_m) or the lag time (Λ). On the contrary, NaCl caused A to increase, with the effect being greatest when the NaCl content was 1.5%. Temperature increase from 10 to 15°C resulted in an approximate doubling of μ_m and unexpectedly an apparent increase in Λ However, this apparent increase resulted from the particular manner in which the lag phase was mathematically calculated.     

Phenotypic characteristics of the three hybridization groups of Aeromonas hydrophila complex isolated from different sources

Biochemical characteristics, SDS-PAGE analysis of whole cell proteins and maximum growth temperature were used for phenotypic characterization of the three DNA hybridization groups (HG 1, HG 2 and HG 3) of the Aeromonas hydrophila complex (96 strains). DNA-DNA hybridization was made with 22 strains using both ³²P- and digoxigenin-labelled probes. The strains originated from raw foods of animal origin, drinking water, fresh water, diseased fish and clinical material. The biochemical tests which were shown to be of value in the separation of the three hybridization groups (HG) were DL-lactate and urocanic acid utilization and acid production from D-sorbitol and from D-rhamnose. A new marker, the maximum growth temperature determined with a temperature-gradient incubator, was shown to separate HG 1 from HG 2 and HG 3. The mean maximum growth temperature ( T _max) of confirmed and putative HG 1 strains was significantly higher (41.1 ±0.56°C; P <0.001>)than that of HG 2 (38.2 ± 0.79°C) or HG 3 (38.6 ± 0.75°C) strains. The lower T _max of HG 2 and HG 3 may explain why the results of biochemical tests may differ if incubation temperatures of 30° or 37°C are used. The SDS-PAGE protein pattern analysis indicated that the strains of HG 3 have a band of variable molecular weight of 24, 25 or 26 kDa not possessed by strains of HG 1 and HG 2. HG 2 strains had two weak bands of molecular weight 24 and 25 kDa. HG 1 organisms were isolated from ground beef, chicken and clinical material but not from fresh water or drinking water. HG 2 and HG 3 organisms were isolated from foods of animal origin and from the water samples.     

Influence of water temperature and feeding regime on otolith growth in Anguilla japonica glass eels and elvers: does otolith growth cease at low temperatures?

The influences of water temperature and feeding regime on otolith growth in Anguilla japonica glass eels and elvers were investigated using individuals reared at 5, 10, 15, 20, 25 and 30° C and in fed or unfed conditions at salinity 32 after their otoliths were marked with alizarin complexone (ALC). To eliminate the difficulty of observing the edges of otoliths with optical (OM) or scanning electron (SEM) microscopes, three to 10 individuals were sampled from each tank at 10, 20 and 30 days during the experiment and reared for an additional 10 days at 25° C after their otoliths were marked a second time. Otolith growth and the number of increments were measured using both OM and SEM. Most A. japonica commenced feeding after 10 days at 20–30° C or after 20 days at 15° C, but no feeding occurred at 5 and 10° C. No otolith growth occurred at 5 and 10° C except in two individuals with minimal increment deposition at 10° C. Otolith growth was proportional to water temperature within 15–25° C and not different between 25 and 30° C. At 15, 25 and 30° C, the mean otolith growth rate in fed conditions was higher than in unfed conditions. The number of increments per day was significantly different among water temperatures (0·00–0·01 day⁻¹ at 5 and 10° C, 0·43–0·48 day⁻¹ at 15° C and 0·94–1·07 day⁻¹ at 20–30° C). These results indicated that otolith growth in A. japonica glass eels and elvers was affected by temperature and ceased at ≤10° C under experimental conditions. Hence, future studies analysing the otoliths of wild-caught A. japonica glass eels and elvers need to carefully consider the water temperatures potentially experienced by the juveniles in the wild.     

Bactericidal effect of chlorine on motile Aeromonas spp. in drinking water supplies and influence of temperature on disinfection efficacy

The susceptibility of toxigenic Aeromonas spp. to free chlorine in drinking water supplies, and the influence of environmental temperature on the bactericidal activity of the oxidant, were evaluated. The results showed inactivation curves characterized by an initial phase of rapid reduction of viable cells followed by a slow inactivation of bacteria. The effect of the chlorine compound was markedly influenced by water temperature. At a summer water temperature (20 °C), the efficacy of the chlorine concentrations tested was found to be two to three times lower compared to that found at a winter temperature (5 °C). Resistance was moderately, but significantly, greater in Aer. hydrophila vs Aer. caviae and Aer. sobria , but all Aeromonas spp. were more susceptible than Escherichia coli . Selective pressure with free chlorine did not produce Aeromonas cells with higher levels of chlorine resistance.     

Growth of Aeromonas spp. at 4°C and related toxin production

Over the last few years Aeromonas spp. have been considered to be potential intestinal pathogens (Altwegg & Geiss 1989). Recently, detailed evidence has linked a strain from contaminated shrimp with a clinical isolate from a patient with diarrhoea and found them to be the same (Altwegg et al. 1991). It is now known that Aeromonas spp. are present in many foods, especially those of animal origin (Palumbo et al. 1989). Furthermore, previous studies have shown that these organisms are quite capable of growing at refrigeration temperatures. For example, Palumbo et al. (1985) found that seven out of 14 strains showed significant growth within 14 d at 5°C. However, most studies have only examined a relatively small number of strains, which were not speciated and were simply called A. hydrophila.
In this paper we examine the growth of 36 strains of aeromonas (divided into A. hydrophila, A. sobria and A. caviae ) at 4°C, and their ability to produce cyotoxins at 4°C, 30°C and 37°C.     

Nitrogen fixation by three species of leguminosae in the Canadian High Arctic Tundra

Abstract. Significant levels of nitrogenase activity (nitrogen fixation) were demonstrated in three species of Arctic legumes ( Oxytropis maydelliana, O. arctobia and Astragalus alpinus ) growing in high tundra at Sarcpa Lake, Melville Peninsula, N.W.T. Nitrogenase activity of intact plants was correlated with the number of nodules per plant, with field soil temperatures and limited by water shortage. Activity in freshly detached nodules showed a plateau of maximum activity between 10°C and 25°C and a near linear decline with temperature down to 0°C. Unusually, the segmented nodules of all three species are perennial in which growth and leghaemoglobin production resumes each spring from an overwintering apical meristem. Nodules are most numerous in the warmer soil stratum (2–10 cm. depth). Other studies indicate that the arctic rhizobia belong to a single cold-adapted species which has co-evolved with the legumes of tundra.     

The influence of pH and temperature on initiation of growth of Salmonella spp.

The ability of 13 strains of Salmonella , representing 12 serotypes, to grow in a tryptone-yeast extract-glucose medium, acidified with HC1 to pH values between 3.80 and 5.60 at intervals of 0.20 units, has been investigated. During incubation at 30°C, growth occurred at minimum pH values of 3.8–4.0 in 1–3 d. At 20°C, growth occurred at minimum pH values of 3.8–4.2 in 3–5 d. In tests incubated at 10°C, growth occurred at minimum pH values of 4.4–4.8 in between 10 and 19 d.     

The effects of phosphate supply on uptake of potassium ions, 2,4-D and atrazine by wheat and maize

The germination percentage of peach [ Prunus persica (L.) Batsch cv. Halford] seeds at 20°C was low (< 20%) after incubation at 5°C for as long as 35 days, but then increased considerably (> 40%) when the seeds were maintained at 5°C for longer than 42 days. Four zones of gibberellin-like activity were found in partially purified seed extracts. Gibberellin-like activity remained low in seeds incubated at 5°C for as long as 28 days, but increased significantly in three of these zones after 35 days, and in the fourth zone after 49 days. The increase in gibberellin-like activity was evident prior to the transfer of the seeds to 20°C. Moreover, seeds maintained at 5°C germinated at this temperature after 63 days. For seeds incubated and germinated at 20°C, both the germination percentage and the gibberellin-like activity remained low throughout the experimental period. Application of the growth retardant paclobutrazol to seeds after 28 days of a 49 day total incubation period at 5°C did not substantially reduce seed germination, although the increase in gibberellin-like activity was prevented. Seeds did, however, require a longer time to germinate after transfer to 20°C and were dwarfed in appearance. Application of GA₃ to seeds prior to stratification increased the percentage germination of seeds only when they had been incubated at 5°C for at least 35 days. The major changes in gibberellin-like activity are, therefore, associated not so much with the processes which allow germination to take place in peach, but more with those processes which allow normal growth and development of the seedling.     

The effects of temperature,water activity and pH on the growth of Aeromonas hydrophila and on its subsequent survival in microcosm water

AIMS: The influence of temperature, water activity and pH on the growth of Aeromonas hydrophila, and on its survival after transfer in nutrient-poor water were assessed. METHODS AND RESULTS: Experiments were carried out according to a Box-Behnken matrix at 10-30 degrees C, 0.95-0.99 water activity (aw) and pH 5-9. The effect of each factor on the kinetic parameters of growth (i.e. the maximal specific growth rate, mumax, and the lag time, lambda) and on the decline of the bacteria in microcosm water (time to obtain a reduction of 5 log, T5 log) were studied by applying central composite design. CONCLUSIONS: The major effect of temperature and water activity on the growth of A. hydrophila was highlighted, whereas the effect of pH in these experimental conditions was not significant. Models describing the effect of environmental parameters on the growth of A. hydrophila were proposed. The effect of the growth environment, and particularly the incubation temperature, have an influence on the survival ability of the bacteria in nutrient-poor water. SIGNIFICANCE AND IMPACT OF THE STUDY: The Box-Behnken design was well suited to determine the influence of environmental factors on the growth of A. hydrophila and to investigate the effect of previous growth conditions on its survival in microcosm water.     

Effect of temperature on life table parameters of Podisus nigrispinus (Het., Pentatomidae) fed with Alabama argillacea (Lep., Noctuidae) larvae

Abstract: The influence of temperature on life table parameters of Podisus nigrispinus (Dallas) (Het., Pentatomidae) fed with Alabama argillacea (Hübner) (Lep., Noctuidae) larvae was studied. This predator was kept at constant temperatures of 20, 23, 25, 28, 30 and 33±0.2°C, at relative humidity of 60±10% and photoperiod of L : D 14 : 10. Gross (GRR) and net ( R ₀) reproductive rates of P. nigrispinus ranged from 1.6 to 366.6 and from 0.02 to 189.5 females/female at temperatures of 33 and 28°C, respectively; generation time ( T  ) ranged from 33.3 (33°C) to 85.5 (20°C) days; doubling time ( D ) from 0.82 (33°C) to 17.8 (20°C) days; intrinsic rate of increase ( r _m ) from −0.13 (33°C) to 0.12 (28°C) per day; and the finite rate of increase ( λ ) from 0.88 (33°C) to 1.12 (28°C) females/female added to the population per day. The ideal age to release P. nigrispinus should be when this predator presents higher reproductive values (VR _x ); that is, its adults are about 7 days old, independent of prevailing temperature. Population growth of P. nigrispinus was affected by temperature with maximum numerical response between 28 and 30°C. The negative population growth shown at 33°C may not occur in natural conditions due to milder microclimate in the cotton agroecosystem and due to oscillations of temperature in the course of the day.     

Effect of pre-incubation temperature on the lag time of Aeromonas hydrophila

The effect of prior incubation temperature (5, 15, 25 and 35°C) on the lag times of two strains of Aeromonas hydrophila at the same four incubation temperatures was assessed. When grown at 5°C the type strain (ATCC 7966) showed a 28-fold difference in lag time depending on whether the inoculum had been incubated at 5 or 35°C. However, the corresponding factor for a food-derived strain was only 4. A similar effect was observed, to a lesser extent, under most of the conditions used. Prior incubation temperature had no effect on generation time.     

Survival, development and food energy partitioning of nase larvae and early juveniles at different temperatures

Survival was generally high, 94–100%, for newly hatched larvae of the nase Chondrostoma nasus held at 10, 13, 16, 19, 22, 25 and 28° C up to day 66 post-fertilization. The developmental rate decreased with age and increased with temperature. Specific growth rates increased with temperature; within one temperature range growth rate decreased with ontogenetic development. Food consumption and respiration increased with temperature and body size. A temperature increase from 25 to 28° C resulted in slightly reduced survival, minor acceleration of developmental growth and respiration rates, and impeded skeleton formation. Growth efficiency of consumed energy decreased throughout the larval period from 55 to 67% at the first larval stage (L1) to 36–48% at the first juvenile stage (J₁). A similar trend for assimilation efficiency and its utilization for growth was observed. The constant temperatures required by larval nase ranged from a minimum 8–10° C to a maximum 25–28° C. A shift of optimum temperatures, 8–12, 13–16, 15–18, 19 and 22° C for nase spawning, embryonic development, yolk feeding larvae, early externally feeding larvae and, late larvae and juveniles, respectively, paralleled the spring rise in the river water temperature. Larval and juvenile nase show high survival, growth and energy conversion efficiencies compared with other fish species. On the other hand, low survival rates and growth can be attributed to external perturbations; thus, young nase may be considered a good indicator of the environmental and ecological integrity of river systems.     

Experimental aflatoxin production in Manchego-type cheese

Manchego-type cheese, a typical Spanish cheese, was inoculated in various ways with an aflatoxigenic organism, Aspergillus parasiticus NRRL 2999, to study the production of aflatoxin. When the original milk was contaminated with a spore suspension, aflatoxin was not detected in paraffin-covered cheeses although it was present in the top layer of non-paraffin-covered cheeses after ripening at 15°C for 60 d. When the cheese surface was inoculated, no aflatoxins were detected in paraffin-covered cheeses after ripening for 60 d although they were found when the cheeses were ripened for 30 d. In non-paraffin-covered cheeses aflatoxins were detected only in the top layer and in the second 10 mm layer when cheeses were incubated after the normal ripening at 28°C for 30 d. When the centre of the cheese was inoculated, no aflatoxins were detected although Aspergillus grew slightly along the inoculation area. When cheese portions were inoculated, fungal growth was evident after incubation at 28° and 15°C for 6 d but there was no growth at 10°C after 50 d. At 28°C aflatoxins were detected at a concentration of 132 μg/g after 13 d, the highest level obtained. In cheese paste at 28° and 15°C, growth was intense, but the level of aflatoxins detected was lower than in cheese portions. At 10°C the growth was heavy, but aflatoxins were not detected.     

Ontogenetic variations of thermal optimum for growth, and its implication on thermolabile sex determination in blue tilapia

Knowledge of how the optimum temperature for growth ( T °_opt) varies during ontogeny, and how close it is to the temperatures that induce phenotypic masculinization is fundamental to the understanding of the evolution of thermolabile sex determinism (TSD) in fishes. In blue tilapia Oreochromis aureus , T °_opt is 32·6° C at the start of exogenous feeding (10mg fish) and it decreases by c . 1° C each time that the fish body mass increases by an order of magnitude. Temperatures <35° C are not sufficient to induce complete phenotypic masculinization. Based on a multiple-regression model ( r ²=0·938) plotting growth against body mass and water temperature, genotypically female tilapia living at high temperatures during the thermosensitive period (21–28 days) and being reversed into phenotypic males would incur an initial growth disadvantage over fish living at T °_opt, but not over those living at slightly colder temperatures (27–29° C). This initial disadvantage would be later compensated for by faster growth because of between-sex growth dimorphism to the detriment of phenotypic females. These arguments suggest that there is no definite pressure against the selection of TSD in blue tilapia and probably other Oreochromis spp.     

Embryonic development, larval growth and life cycle of Coenagrion puella (Odonata: Zygoptera) from an Austrian pond

SUMMARY. 1. Newly-laid eggs of Coenagrion puella (L.) from a pond near Herzogenburg (Lower Austria) were kept at constant water temperatures (range c .3.5°C to c .28°C)in the laboratory. Hatching success varied with temperature; no eggs hatched below 12°C and nearly all hatched at c .l6°C. Hatching time decreased with increasing temperature and the relationship between the two variables within the range 12–28 °C was well described by a power law. The length of the hatching period was less than 12 days. Hatching times estimated from the power-law equations and those obtained in the field experiments were similar. Therefore both the hatching time and the length of the hatching period in the field could be estimated from the laboratory data for the range 12–28°C.
2. The maximum number of instars from egg to imago was 11; the average body length increment (mm) per moult was proportionately constant at c .26% and Dyar's rule was applicable. The interval between moults decreased with increasing temperature up to the seventh instar and the relationship between the two variables within the range 12–28°C was well described by a power law. The moulting interval for instars 8–11 ranged from 23 to 48 days and was relatively independent of temperature. No moulting occurred at temperatures below 12°C.
3. Larval growth was logistic in the laboratory and variations in mean logistic growth rate (range 0–2.5% length day⁻¹) were related to mean temperature with no growth at temperatures <12°C. Larval growth rates in pond experiments were similar to those estimated from laboratory data, and therefore the regression equations obtained from the laboratory experiments are probably applicable to larval growth in the field.
4. Information on the life cycle of C. puella is briefly reviewed and it is concluded that C. puella from the pond near Herzogenburg has an univoltine life cycle.