Transformation of Streptococcus bovis protoplasts by plasmid DNA

M. MAREKOVÁ, V. KMET' AND P. JAVORSKÝ. 1996. The transformation and subsequent regeneration of ruminal strain Streptococcus bovis AO24/85 protoplasts by plasmid DNA was studied. The best stabilizer for regeneration of protoplasted cells was 5% sucrose in the regeneration medium and in the agar plates. Optimal concentration of polyethylene glycol 6000 in the transformation medium was 25% for both plasmids tested. Addition of Ca²⁺ and Mg²⁺ ions (2.5 mmol l^-1) to the transformation medium increased the proportion of regenerated cells. Transformation frequencies were 3 times 10³ transformants per μg of pNZ12 and 2.4 times 10² per μg of pJK108, respectively.     

Purification and partial characterization of a toxic serine protease produced by pathogenic Vibrio alginolyticus

An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn (Penaeus japonicus) was purified using the AKTA purifier system with hydrophobic interaction chromatography, anion exchange and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl methylsulphonyl fluoride (PMSF), antipain and shows maximal activity at pH 8 to 11, having a pI of 4.3 and a molecular weight of approximately 33 kD. The toxin was completely inhibited by FeCl2 but partially inhibited by 3,4-dichloroisocoumarin (3,4-DCI), ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), CuCl2 and ZnCl2. The purified protease was lethal for kuruma prawn at an LD50 of 0.29 microgram protein/g body weight. The haemolymph withdrawn from the moribund prawns injected with the toxic protease was unable to clot. The coagulogen in the kuruma prawn plasma showed an increased migration rate after incubation with this serine protease, and a plasma colour change from blue to pink was recorded. The addition of PMSF completely inhibited the lethal toxicity of the purified protease, indicating that this serine protease was a lethal toxin produced by the bacterium. The 33 kD protease was therefore a toxic protease produced by V. alginolyticus strain Swy.     

The effect of moisture potential on growth and survival of root nodule bacteria in peat culture and on seed

Peat from three sources was dried, milled and packed separately in polyethylene bags and sterilized by irradiation. The carrier was impregnated with broth cultures of either Rhizobium leguminosarum bv. trifolii strain WU95, Bradyrhizobium japonicum strain CB1809 or B. lupini strain WU425 and sterile water to provide five moisture potentials in the range > - 1 × 10⁴ - 1 × 10⁶ Pa. The packets were stored at 26°C under conditions which restricted moisture loss. Numbers of root nodule bacteria were counted at intervals up to 12 weeks. No single moisture potential was optimum for all strains in all carriers because of a significant ( P < 0.05) interaction between moisture potential × strain × carrier × time. Where direct comparisons could be made, all strains survived best at - 1 × 10⁴ and/or −3.2 × 10⁴ Pa. Seeds of Trifolium subterraneum and polypropylene beads (used to avoid seed coat toxicity), were inoculated with WU95 prepared in two sources of peat and at each of the above moisture potentials and stored at 15°C. Seed coat toxicity significantly effected the log death rate ( k ) of WU95 on subterraneum clover seed for the period 0–0.25 d ( k 1.796) compared with k - 0.399 for polypropylene beads. In the first 24 h moisture did not affect survival but by 28 d rhizobia grown in Badenoch peat survived best at −3.2 × 10⁴ Pa. In Millicent peat, survival was equally as good at −3.2 × 10⁴ and −1 × 10⁴ Pa.     

The use of direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) to assess microbial populations on food contact surfaces

Two rapid methods, direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) on swab resuspension fluids, were compared with the traditional total viable count (TVC) on swab resuspension fluids for their ability to enumerate surface populations of attached bacteria. The degree of error in estimating surface populations was shown to be significantly less with DEM than DEFT followed by TVC. DEM estimated populations in the range 3 times 10³ to 5 times 10⁷ colonies/cm² whilst DEFT enumerated populations above 3 times 10⁴ colonies/cm² and TVC above 3 times 10⁵ colonies/cm² (as measured by DEM). Swabbing was shown to remove a constant proportion of organisms from the surface populations tested, although below 3 times 10⁵ colonies/cm² most of the organisms remained in the cotton matrix and were difficult to resuspend. DEFT was more able to enumerate swab resuspension fluids obtained from surface populations below 3 times 10⁵ colonies/cm² than was TVC.     

Fate of Listeria monocytogenes during manufacture and ripening of semi-hard cheese

Semi-hard cheeses were experimentally elaborated with pasteurized milk from sheep, goat and cow (15: 35: 50) and inoculated to contain 1.9 times 10⁵ Listeria monocytogenes /ml in cheeses 1 and 2 and 4 times 10³ L. monocytogenes /ml in cheeses 3 and 4. Counts of L. monocytogenes were determined by direct surface plating of samples on listeria selective agar medium. The results show the substantial survival of L. monocytogenes present in milk during manufacture and ripening of this type of cheese.     

Pathogenicity of different isolates of Vibrio harveyi in tiger prawn, Penaeus monodon

P.-C. LIU, K.-K. LEE AND S.-N. CHEN. 1996. The pathogenicity of six Vibrio harveyi strains in tiger prawn, Penaeus monodon , was studied, using both live bacteria and extracellular products (ECP). The organisms originally isolated from diseased penaeids were more virulent using both live bacteria and ECP (LD₅₀, 4.87–8.65 times 10⁴colony-forming units (cfu) and 1.20–1.51 μg protein g^-1body weight) than the two reference strains originally isolated from either sea water (ATCC 25919; LD₅₀, 3.18 times 10⁶cfu and 2.70 μg protein g^-1body weight) or diseased Talorchestia sp. (ATCC 14126, 0.418 times 10⁶cfu and 2.34 μg protein g^-1body weight). Each strain was reisolated from the haemolymph and the hepatopancreas of moribund prawns following each bacterial challenge. Both the live bacteria and the ECPs of the penaeid isolates exhibited stronger proteolytic (caseinase), phospholipase and haemolytic activities than those of the reference strains. These results indicate that there are differences between penaeid and non-penaeid isolates of V. harveyi in pathogenicity and reveal that proteases, phospholipases, haemolysins or exotoxins might play leading roles in the pathogenicity of V. harveyi in the tiger prawn, Penaeus monodon .     

Lethal attribute of serine protease secreted by Vibrio alginolyticus strains in kuruma prawn Penaeus japonicus

Toxicity of the extracellular products (ECP) and the lethal attribute of serine protease secreted by five pathogenic Vibrio alginolyticus strains from various sources in kuruma prawn Penaeus japonicus were studied. The ECPs of organisms originally isolated from diseased kuruma prawn or small abalone Haliotis diversicolor supertexta were more lethal (LD50 value of 0.48 or 0.41 microg protein/g prawn) than those from diseased tiger prawn P. monodon, yellowfin porgy Acanthopagrus latus or horse mackerel (LD50 value of 0.98-1.17 microg protein/g prawn). All the ECPs manifested strong, weak and no activities against gelatin, sheep erythrocytes and chitin, respectively. In immunodiffusion tests using rabbit antiserum to a purified 33 kDa serine protease of strain Swy against ECP of each tested strain produced one single precipitation band in each treatment. Furthermore, the serine protease was suggested to be the dominant protease secreted by V. alginolyticus strains tested since the majority of enzymatic activity of the respective ECP was inhibited by phenylmethanesulfonyl fluoride (PMSF). A higher inhibition of serine protease activity by PMSF resulted in lower mortality rate of the ECPs injected into the prawns suggesting that the protease is one of the major lethal factor(s) secreted by V. alginolyticus.     

Distribution of Synechococcus spp. determined by immunofluorescent assay

By using two polyclonal antisera against WH 7803 strain (Synechococcus sp.) and WH 5701 strain (Synechococcus bacillaris) it is possible to detect and to enumerate cells of the two cyanobacterial serogroups. The immunofluorescence technique was used to study the distribution of the two serogroups in the estuarine, coastal and upwelling waters of the Mediterranean Sea surrounding Messina. In the estuarine waters of the Alcantara River (Ionian Sea), the WH 7803 serogroup was present at a concentration in the order of 10² cells ml⁻¹ and the WH 5701 serogroup at a concentration of 5·5 × 10² cellsml⁻¹. In the coastal waters of Messina, where urban and industrial wastes are usuallydumped, the concentration of total phycoerythrin- Synechococcus ranged from 1·3 × 10² to 4·1 × 10³ cells ml⁻¹; the WH 7803 serogroup accounted for 50–94% of the totalpopulation in Ionian stations, whereas the WH 5701 serogroup ranged from1·4 × 10¹ to6·7 × 102cells ml⁻¹. In the upwelling area (Straits of Messina) bothserogroups were found. Vertical distribution of two Synechococcus strains had anopposite trend and their concentrations were of the order of 10¹–10²cells ml⁻¹. Theuse of the Scan laser system allows both autofluorescent and labelled organismsto be distinguished in a preparation for optical microscopy. It also allows false-positivecells to be distinguished.     

Effect of rigidity of gel medium on benzyladenine-induced adventitious bud formation and vitrification in vitro in Picea abies

Observations on the effects of different degrees of rigidity of both an agar (Tayio) and a non-agar (Gelrite) gel on the uptake of radiolabelled N⁶-benzyladenine (¹⁴C-BA) were also extended to mode of application and positioning of the explant. Regression analysis showed a highly significant inverse correlation between ¹⁴C-BA accumulation and degree of gel stiffness. Significantly greater numbers of adventitious buds per explant were induced at low to medium levels of rigidity (2.5–10 g Tayio 1⁻¹, 1–5 g Gelrite 1⁻¹); this advantage was almost completely nullified at the lower levels (2.5 and 5.0 g Tayio 1⁻¹, 1 and 1.5 g Gelrite 1⁻¹) as a result of the high incidence of vitrification. In addition to turgor distension, vitrified buds displayed cellular damage. Explants with their cotyledons flattened onto the agar surface accumulated less ¹⁴C-BA after 96 h than upright explants, but produced greater numbers of adventitious buds, pseudobuds and phylloids. It was suggested that BA was taken up only by "target" cells, presumably the differentiating subsidiary cells of those stomatal complexes in surface contact with the medium. Pulse treatments of relatively short durations (2 h) with optimal concentrations of BA (ca 125 μ M ), followed by subculturing on hormone-free media gelled with 10 g agar 1⁻¹, produced a satisfactory balance between yield and competence of adventitiously-induced buds.     

Longevity of the freshwater anostracan Streptocephalus mackini (Crustacean: Anostraca) in relation to food (Chlorella vulgaris) concentration

SUMMARY 1. Temporary ponds are inhabited by a variety of invertebrates, of which anostracans are an important group. We studied the lifetables of male and female anostracan Streptocephalus mackini at 3 algal concentrations (0.5 × 10⁶, 1.0 × 10⁶ and 1.5 × 10⁶ cells mL⁻¹).
2. Regardless of sex, S. mackini showed better survivorship at lower food levels. The longest average lifespan observed was 85 ± 2 days for males fed Chlorella at 0.5 × 10⁶ cells mL⁻¹.
3. Both net reproductive rate and generation time decreased with increasing food level. The highest net reproductive rate was about 120 cysts per female. The longest generation time of about 40 days, observed at 0.5 × 10⁶ cells mL⁻¹, was more than three times that at 1.5 × 10⁶ cells mL⁻¹.
4. The rate of population increase ( r ) was nearly the same (0.31 ± 0.06) at high (1.5 × 10⁶ cells mL⁻¹) and intermediate (1.0 × 10⁶ cells mL⁻¹) food levels. The r -value at low food level (0.5 × 10⁶ cells mL⁻¹ of Chlorella ) was 0.20 ± 0.01 per day.     

Monitoring the fate of genetically engineered bacteria sprayed on the phylloplane of bush beans and grass

Abstract The fate of a Bacillus amyloliquefaciens with the recombinant plasmid pSB20 sprayed on the phyllosphere of grass, and of a Tn 5 marked Pseudomonas syringae sprayed on the phyllosphere of bush beans was studied in planted soil microcosms. B. amyloliquefaciens showed a decline from 1.5×10⁸ to 3.1×10² cfu g⁻¹ on the phylloplane of grass in the course of the experiment. B. amyloliquefaciens was easy to follow by selective cultivation due to the complete absence of bacterial background growth. Southern blot hybridization of Hin dIII digested genomic DNA showed plasmid restriction patterns identical with pSB20 indicating high plasmid stability. In total DNA extracts from phyllosphere bacteria the recombinant plasmid was detectable by Southern blot hybridization up to 6×10⁴ cfu g⁻¹ (wet weight). Counts of hybridizing colonies showed that P. syringae established on the phyllosphere of bush beans at between 5×10³ and 4×10⁶ cfu g⁻¹ fresh weight. During senescence of the bean plants the strain was no longer detectable by selective cultivation and subsequent colony hybridization. In contrast, Tn5 marked DNA was detected after PCR amplification over the whole period of the experiment.     

Unmodified and recombinant strains of Lactobacillus plantarum are rapidly lost from the rumen by protozoal predation

A genetically-manipulated strain of Lactobacillus plantarum and the unmodified parent strain were introduced into the rumen of sheep at an initial inoculum level of 1 times 10⁷ cfu ml^-1 of rumen fluid. There were no significant differences between the viable counts of the two inoculants throughout a 24 h sampling period. The rates of loss were 0.36 and 0.29 h^-1 (proportion of colony-forming units lost, measured over the first 2 h) for the parent strain and recombinant strain respectively, and within 24 h of inoculation neither of the strains were detectable in rumen fluid. Further experiments in vitro revealed that the inoculants persisted in sterile rumen fluid with a loss rate of 0.044 and 0.057 h^-1 for the parent strain and the recombinant strain respectively. Incubations with rumen fluid alone, protozoa-free rumen fluid and protozoa-enriched rumen fluid revealed that protozoal predation was the most significant factor in the loss of the introduced population. The loss rates from protozoa-free rumen fluid were not significantly different (P < 0.05) from those observed in sterile rumen fluid.     

Food intake and growth in the Moses perch, Lutjanus russelli (Bleeker), with reference to predation on penaeid prawns

Juvenile Moses perch were fed various rations of penaeid prawn and pilchard foods at 26.4 and 28.7° C to elucidate feeding–growth relationships. Maintenance rations amounted to 5.0 and 3.8% of body weight per day (BW day^–1) on prawn and pilchard diets, respectively, at 26.4° C and 4.5% BW day ¹ for prawn at 28.7° C. This apparent reduction in maintenance ration at higher temperature is probably due to reduction in activity levels. Starvation weight loss, an indicator of resting metabolic rate, increased with temperature and exhibited a Q¹⁰ of 2.0 to 2.4.
Fish fed ad libitum once or twice per day at 26.4° C grew at 1.1 % BW day^–1 and ingested about 9% of their BW day^–1. At 28.7° C fish ingested about 8% of their BW day^–1 and grew 1 .0% BW day^–1. These growth rates are comparable to those of similar sized lutjanids in the wild. Since prawns comprise 64% of the diet of Moses perch in northern Australia, this level of consumption would require about 6% BW day^–1 of prawns. The relevance of these findings to the assessment of predator impact on prawns in Australian estuaries is discussed.     

Survival of κ-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Abstract A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty-two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g⁻¹ of dry soil based on the polymerase chain reaction (PCR). Survival of κ-carrageenan-encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence-based nonselective plating and PCR-amplification of a tnsA fragment. After freeze-thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 10⁹ cfu g⁻¹ of dry soil to below the detection threshold of both selective (14 cfu g⁻¹ of dry soil) and nonselective (1 × 10³ cfu g⁻¹ of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 10⁶ to 2.9 × 10⁸ cfu g⁻¹ of dry soil after a 3-week incubation at 22°C and declined to 6.3 × 10⁶ cfu g⁻¹ of dry soil after the freeze-thaw treatment.     

Microbial load in poultry feed and detection of aflatoxin B1 using monoclonal antibody-based enzyme linked immunosorbent assay

Feed samples collected from different poultry farms and feed mills situated in Andaman and Nicobar islands in India were assessed for microflora and aflatoxin B₁ contamination. The bacterial counts ranged from 1.0 times 10⁷ to 8.8 times 10⁷ cfu/g of the feeds, while counts of fungi ranged from 1.0 times 10³ to 8.7 times 10³ cfu/g. The mycoflora comprised mainly of Aspergillus spp., A. flavus being most dominant. Aflatoxin B₁ was detected by monoclonal antibody-based enzyme linked immunosorbent assay technique and the content in different feed samples ranged from 5.5 to 90 ng/g.     

The relationship between cytotoxic effect and binding to mammalian cultured cells of Clostridium perfringens enterotoxin

Abstract The relationship between the cytotoxic effect and binding to different cell lines of Clostridium perfringens enterotoxin was investigated. The enterotoxin released ⁵¹Cr from Vero and MDCK cells labeled with Na₂-⁵¹CrO₄. The effect varied depending upon the dose of enterotoxin and the duration and temperature of the interaction. The enterotoxin gave no effect on FL, KB, or L-929 cells. [¹²⁵I]Enterotoxin bound specifically to Vero and MDCK cells via a binding site of distinct nature, but not to FL, KB, or L-929 cells. The number of the binding sites located on one MDCK cell (1.98 × 10⁶ sites/cell) was three times that on one Vero cell (5.64 × 10⁵ sites/cell), although the binding affinity of MDCK cell ( K _a/ 3.76 × 10⁷ M⁻¹) was 0.1 that of Vero cells ( K _a/ 3.23 × 10⁸ M⁻¹). Binding of the enterotoxin to susceptible cells was temperature-independent.     

Molecular Detection of Phytophthora cryptogea on Calendula officinalis and Gerbera jamesonii Artificially Inoculated with Zoospores

In this work, a protocol for zoospores production of Phytophthora cryptogea , an economically important plant pathogen was optimized. Five different concentrations of zoospores (5 × 10⁵, 5 × 10⁴, 5 × 10³, 5 × 10², 5 × 10¹ zoospores/ml) from four different isolates of P. cryptogea (Maria 1, Maria 2, S3 1-A, Amazzone) were used as inoculum on pot marigold ( Calendula officinalis ) and gerbera ( Gerbera jamesonii ) plants. Maria 1 was the most virulent isolate both on pot marigold and gerbera plants according to disease severity. A rapid and sensitive pathogen DNA extraction protocol suitable for large quantities of plant samples was adopted. Conventional polymerase chain reaction (PCR) was able to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 12) and gerbera plants (day 8) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴, 5 × 10³ P. cryptogea  zoospores/ml. Real-time PCR showed the possibility to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 8) and gerbera plants (day 4) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴ P. cryptogea  zoospores/ml. The first symptoms appeared on pot marigold plants 14 days after pathogen inoculation and on gerbera plants 10 days after inoculation. Real-time PCR showed the possibility to detect the pathogen 4 days before conventional PCR and 6 days before the appearance of disease symptoms both on pot marigold and gerbera plants.     

Improved PCR detection sensitivity of Erwinia carotovora subsp. atroseptica in potato tuber peel extract by prior enrichment on a selective medium

A simple and sensitive method was developed to replace the need for complex and laborious DNA extraction to remove inhibitory substances in potato tuber peel extract before detection of Erwinia carotovora subsp. atroseptica (Eca) by PCR. Eca was enriched by a factor of 10⁵ when peel extract was inoculated onto a selective medium, CVP, and incubated at 27°C for 24 h. Bacterial micro-colonies which developed were suspended in 500 μl of water and the bacteria diluted in water 100-fold, or 10-fold followed by washing by centrifugation, before PCR testing. The sensitivity of detection obtained with the former was ca 10¹–10² cells ml⁻¹ and with the latter ca 10¹ cells ml⁻¹, when different numbers of streptomycin-resistant Eca strain were added to peel extract from three Eca-free potato cultivars. The method was validated and the sensitivity confirmed relative to two different commonly used Eca detection methods using naturally contaminated tubers.     

Sodium Transport from Blood to Brain: Inhibition by Furosemide and Amiloride

Abstract: Brain sodium uptake in vivo was studied using a modified intracarotid bolus injection technique in which the uptake of ²²Na ⁺ was compared with that of the relatively impermeable molecule, [³H]l-glucose. At a Na ⁺ concentration of 1.4 m M , Na ⁺ uptake was 1.74 ± 0.07 times greater than l -glucose uptake. This decreased to 1.34 ± 0.04 at 140 m M Na ⁺, indicating saturable Na ⁺ uptake. Relative Na ⁺ extraction was not affected by pH but was inhibited by amiloride ( K _i= 3 ± 10⁻⁷ M ) and by 1 m M furosemide. The effects of these two inhibitors were additive. Brain uptake of ⁸⁶Rb ⁺, a K ⁺ analogue, was measured to study interaction of K ⁺ with Na ⁺ transport systems. Relative ⁸⁶Rb ⁺ extraction was also inhibited by amiloride; however, it was not inhibited by furosemide. The results suggest the presence of two distinct transport systems that allow Na ⁺ to cross the luminal membrane of the brain capillary endothelial cell. These transport systems could play an important role in the movement of Na ⁺ from blood to brain.     

Polymerase chain reaction detection and speciation of Campylobacter upsaliensis and C. helveticus in human faeces and comparison with culture techniques

A polymerase chain reaction (PCR) assay based on the 16S rRNA gene and an improved DNA extraction procedure were developed for the direct detection and differentiation of Campylobacter upsaliensis and C. helveticus in seeded human faeces. The PCR assay was compared with culture detection by a membrane filter (MF) technique and on selective agar (SA) containing 8 mg l⁻¹ cefoperazone. Both MF culture and the PCR assay detected 10⁵ colony-forming units (cfu) g⁻¹ faeces. Selective agar culture of some strains could detect as few as 10³ cfu g⁻¹ faeces. However, some strains were susceptible to cefoperazone and either failed to grow or were detected only with reduced sensitivity in the presence of the antibiotic. Detection by MF and SA both required 48–96 h incubation in a microaerobic atmosphere and did not specifically identify the isolate. By contrast, the PCR assay could be completed within 8 h and accurately identified the two phenotypically similar species, C. upsaliensis and C. helveticus.