Elevated temperature method for recovery of Vibrio cholerae from oysters (Crassostrea gigas)

Of 222 Vibrio cholerae isolates from diverse clinical and environmental sources, 219 produced visible growth in alkaline peptone broth when incubated overnight at 42 degrees C. In field trials conducted to compare enrichment at incubation temperatures of 42 and 35 degrees C, significantly higher rates of isolation (P less than 0.05) and recovery (P less than 0.01) of V. cholerae from oysters were observed at 42 degrees C.     

Regulation of toxin biosynthesis by plasmids in Vibrio cholerae

Vibrio cholerae strain 569B Inaba harbouring P plasmid produced less toxin than the parent strain. To examine the effect of plasmid loss on toxin production, temperature-sensitive (ts) mutants of P, unable to replicate at 42 degrees C, were isolated. One ts plasmid was unstable at 42 degrees C and its loss yielded a cured strain that resumed a normal level of toxin biosynthesis characteristic of the plasmid-free parent strain. Toxin production was again suppressed in the cured strain after reacquisition of P plasmid. This suggested a role for plasmid-borne genes in the regulation of toxin biosynthesis. A mutant of strain 569B Inaba that produced mutant toxin was isolated by transfer of P and V plasmids. The mutant toxin was similar to choleragenoid because it did not give rise to symptoms of cholera but induced antitoxin immunity in rabbits.     

Prostaglandin secretion by endometrium of pregnant and cyclic cattle at day 17 after oestrus in response to in-vitro heat stress

Bilateral perifusion devices were utilized to measure prostaglandin F-2 alpha (PGF) secretion by bovine endometrium in response to in-vitro heat stress. Tissues were collected at Day 17 after oestrus from cyclic (N = 4) and pregnant (N = 5) cows, placed into 3 perifusion devices, perifused (3 ml/10 min, Krebs-Ringer-bicarbonate [KRB]) for 5 h, and fractions were collected every 10 min. Endometrial tissues within each device were subjected to a different temperature and oxytocin (1 i.u./ml KRB) treatment sequence: (1) control-oxytocin: 1 h at 39 degrees C; 2 h at 39 degrees C, 0.5 h at 39 degrees C with oxytocin, 0.5 h at 39 degrees C and 1 h at 39 degrees C; (2) heat-oxytocin: 1 h at 39 degrees C, 2 h at 42 degrees C, 0.5 h at 42 degrees C with oxytocin, 0.5 h at 42 degrees C and 1 h at 39 degrees C; (3) heat-KRB: 1 h at 39 degrees C, 2 h at 42 degrees C, 0.5 h at 42 degrees C, 0.5 h at 42 degrees C and 1 h at 39 degrees C. Regardless of reproductive status, heat stress induced a rapid increase (P less than 0.01) in PGF secretion rates. Oxytocin induced an increase (P less than 0.01) in PGF secretion for endometrium from cyclic cows regardless of temperature. Endometria from pregnant cows did not respond to oxytocin when perifused at 39 degrees C. However, PGF secretion rates from endometrium of pregnant cows increased (P less than 0.01) in response to oxytocin when perifused under heat stress conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arterial vs. rectal temperature in ponies: rest, exercise, CO2 inhalation, and thermal stresses

We assessed in ponies the adequacy of using rectal (Tre) rather than arterial temperature (Tar) under conditions common to ventilatory control experiments, i.e., CO2 breathing, thermal stress, and particularly exercise. We were interested in whether, and to what extent, Tar-Tre differences could lead to errors in arterial blood gas corrections. At control environmental temperatures (Ta) of 5 degrees C in the winter and 21 degrees C in the summer, Tar and Tre (37.1 degrees C) did not differ (P greater than 0.05). Elevating winter or summer Ta by 10-18 degrees C for 2-days or lowering summer Ta by 9 degrees C (2-days) did not change Tar or Tre (P greater than 0.05). Furthermore, elevating inspired PCO2 to 42 Torr for 15 min did not alter Tar or Tre from control (P greater than 0.05). During treadmill exercise, at 1.8 mph 5% grade, Tar and Tre did not change significantly (P greater than 0.05) from rest by 11 min of work. At 3 mph 5% grade, Tar increased progressively by 0.3 degrees C (P less than 0.05) while Tre tended to increase 0.1 degree C by 11 min. During moderate exercise at 6 mph 5% grade, Tar increased 0.9 degree C (P less than 0.05) while Tre increased 0.25 degree C (P less than 0.05). Finally, by 6 min of heavy exercise at 8 mph 20% grade, Tar increased 2 degrees C (P less than 0.05) while Tre increased 0.5 degree C (P less than 0.05). The Tar-Tre differences during the latter three work loads were statistically significant (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Maintenance of broad host range vector pKT230 in marine unicellular cyanobacteria

Clinical isolate of Vibrio mimicus were examined for production of cell-associated hemagglutinin (HA) and pili and for adherence to formalin-fixed human intestinal mucosa. V. mimicus grown on CFA agar for 3 h at 37 degrees C possessed HA and adhered better to the mucus layer than to the epithelial cell surface. A significant correlation was found between the HA titers and adherence ability to the epithelial cell surface of villi (P less than 0.05); adherence to the ileal lymphoid follicle-associated epithelium occurred at higher levels. In contrast, V. mimicus grown on CFA agar for 20 h at 37 degrees C exhibited lower levels of HA and reduced adherence ability. The production of pili was more pronounced after 20 h of incubation than after 3 h of incubation. In comparison with V. cholerae 01 and V. cholerae non-01 cultured under similar conditions, V. mimicus showed inferior adherence, but with similar HA production or piliation.     

Effect of in-vitro heat stress on prostaglandin and protein secretion by endometrium from pregnant and cyclic gilts at day 14 after oestrus

Endometrium from cyclic (N = 4) and pregnant (N = 4) gilts at Day 14 after oestrus was placed into three bilateral perifusion devices which allow separate perifusion of luminal and myometrial sides. Perifused endometrium was subjected to 39 or 42 degrees C. Incorporation of [3H]leucine into secreted and tissue proteins by endometrial explants following incubation at 39 or 42 degrees C was examined using trichloroacetic acid (TCA) precipitation and one-dimensional SDS polyacrylamide gel electrophoresis. Secretion of PGF was greater from the myometrial side for cyclic gilts (endocrine orientation), but greater from the luminal side for pregnant gilts (exocrine orientation). Regardless of reproductive status or endometrial side, heat stress induced a rapid increase (P less than 0.01) in PGF secretion rates. However, PGF secretion in response to heat stress was greater (P less than 0.01) from the myometrial side and greater (P less than 0.01) for pregnant gilts. PGF secretion rates increased by 63% and 42% from the luminal side, and 40% and 156% from the myometrial side in response to heat stress for cyclic and pregnant gilts, respectively (status x treatment x side interaction; P less than 0.01). Heat stress did not alter incorporation of [3H]leucine into secreted proteins regardless of reproductive status, while incorporation into tissue proteins was decreased (P less than 0.05) by heat stress for pregnant gilts, but not altered for cyclic gilts. Heat stress, in vitro, redirects PGF secretion for endometria of pregnant gilts from an exocrine to an endocrine orientation where it would be available to effect luteolysis and compromise the establishment of pregnancy.     

Seasonal distribution of facultatively enteropathogenic vibrios (Vibrio cholerae, Vibrio mimicus, Vibrio parahaemolyticus) in the freshwater of the Elbe River at Hamburg

Between June 1981 and December 1982 the incidence of Vibrio cholerae, V. mimicus and V. parahaemolyticus was determined at two sampling sites on the Elbe River at Hamburg. A total of 183 strains was isolated from 147 water samples. Of these, 107 belonged to non-01 V. cholerae (ten strains producing a cholera-like enterotoxin); 33 were identified as V. mimicus, including two enterotoxin producers; 42 strains were Kanagawa-negative cultures of V. parahaemolyticus; and one was V. fluvialis. The highest incidence was observed from June to September with about 10(2) organisms/l. Halophilic vibrios, less than five organisms/l, were detectable during the period June/July to October. The vibrio incidence was not influenced by the numbers of aerobic heterotrophic bacteria, coliforms or faecal bacteria. In general water temperature correlated with the seasonal variation. Thus, a temperature rise over 10 degrees to 20 degrees C was followed by a distinct increase in vibrio numbers. Of 14 chemical parameters only chloride concentration might have had an influence on the seasonal variation. It is concluded that the three Vibrio species are indigenous organisms of the Elbe River.     

Chloroplastic NAD(P)H dehydrogenase in tobacco leaves functions in alleviation of oxidative damage caused by temperature stress

In this study, the function of the NAD(P)H dehydrogenase (NDH)-dependent pathway in suppressing the accumulation of reactive oxygen species in chloroplasts was investigated. Hydrogen peroxide accumulated in the leaves of tobacco (Nicotiana tabacum) defective in ndhC-ndhK-ndhJ (DeltandhCKJ) at 42 degrees C and 4 degrees C, and in that of wild-type leaves at 4 degrees C. The maximum quantum efficiency of PSII decreased to a similar extent in both strains at 42 degrees C, while it decreased more evidently in DeltandhCKJ at 4 degrees C. The parameters linked to CO(2) assimilation, such as the photochemical efficiency of PSII, the decrease of nonphotochemical quenching following the initial rise, and the photosynthetic O(2) evolution, were inhibited more significantly in DeltandhCKJ than in wild type at 42 degrees C and were seriously inhibited in both strains at 4 degrees C. While cyclic electron flow around PSI mediated by NDH was remarkably enhanced at 42 degrees C and suppressed at 4 degrees C. The proton gradient across the thylakoid membranes and light-dependent ATP synthesis were higher in wild type than in DeltandhCKJ at either 25 degrees C or 42 degrees C, but were barely formed at 4 degrees C. Based on these results, we suggest that cyclic photophosphorylation via the NDH pathway might play an important role in regulation of CO(2) assimilation under heat-stressed condition but is less important under chilling-stressed condition, thus optimizing the photosynthetic electron transport and reducing the generation of reactive oxygen species.     

Effects of hyperthermia on plasma glycoprotein catabolism by the isolated perfused rat liver

Clearance and degradation of the glycoprotein, asialofetuin (AF), by the isolated perfused rat liver at supranormal temperatures were investigated. The half-life for disappearance of AF was similar at 37, 41, and 42 degrees C, P greater than 0.05. There was a significant difference between the amount of hydrolysis of AF at 37, 41, and 42 degrees C, P less than 0.05. This indicates that there was significant retardation of lysosomal proteolysis or receptor endocytosis by the hepatocyte at elevated temperatures.     

The cellular regulation of vesicle exocytosis by Entamoeba histolytica

We studied the cellular regulation of vesicle exocytosis by Entamoeba histolytica utilizing release of endocytosed 125iodine (125I) labeled tyrosine conjugated dextran; 125I-dextran entered the acid pH vesicles of the amebae and was not degraded during these studies. Exocytosis was temperature dependent with 74%, 36%, 4%, and 0% of 125I-dextran released after 120 min at 37 degrees C, 31 degrees C, 25 degrees C, and 4 degrees C, respectively (P less than 0.01 for each). Exocytosis at 37 degrees C was inhibited by cytochalasin D (10 micrograms/ml), EDTA (10 mM), or the putative intracellular calcium antagonist TMB-8 (250 microM) (P less than 0.01 for each at greater than or equal to 60 min). Calcium ionophore A23187 (1 microM) enhanced exocytosis at 5 and 15 min (P less than 0.01). Elevation of vesicle pH with NH4Cl (10 mM) had no effect on release of 125I-dextran; phorbol myristate acetate (10(-6) M) increased exocytosis by 46% at 30 min (P less than 0.01). Centrifugation of amebae with target Chinese hamster ovary cells resulted in decreased 125I-dextran release into the cell supernatant after 30 and 60 min at 37 degrees C (by 40% and 42%, respectively, P less than 0.01); release of 125I-dextran returned to control values with addition of 1.0 g% galactose or GalNac but not with mannose or N-acetyl-D-glucosamine. Amebic phagocytosis of serum-exposed latex beads had no effect on release of dextran by amebae (n = 16). Exocytosis of acid pH vesicles by E. histolytica is temperature-, microfilament-, and calcium-dependent, and stimulated by phorbol esters.     

Thermal adjustments to nonexertional heat stress in mature and senescent Fischer 344 rats

The purpose of this study was to test the hypothesis that the rise in colonic temperature (Tc) during nonexertional heat stress is exaggerated in senescent (SEN, 24 mo, n = 12) vs. mature (MAT, 12 mo, n = 15) conscious unrestrained Fischer 344 rats. On 2 separate days (48 h apart) each SEN and MAT animal was exposed to an ambient temperature (Ta) of 42 degrees C (relative humidity 20%) until a Tc of 41 degrees C was attained and then cooled at a Ta of 26 degrees C until Tc returned to the initial control level. Control Tc was similar in the two groups for both trials. The rate of Tc change during heating was 63% greater (0.070 +/- 0.005 vs. 0.043 +/- 0.004 degrees C/min, P less than 0.05) and the time to 41 degrees C reduced by 36% (54 +/- 6 vs. 85 +/- 10 min, P less than 0.05) in MAT vs. SEN animals during the first exposure, although the cooling rate was slower in the MAT (0.048 +/- 0.004 degrees C/min) vs. SEN (0.062 +/- 0.006 degrees C/min) animals (P less than 0.05). The heating rate was unchanged in MAT animals between trials 1 and 2. However, SEN animals had a 95% increase in heating rate in trial 2 compared with trial 1 (P less than 0.05), and the corresponding time to 41 degrees C was decreased by 44% (P less than 0.05). As a result, rate of heating and time to 41 degrees C were similar in the two groups during trial 2. The cooling rate was similar between trials within each group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism for pressor response to nonexertional heating in the conscious rat

The purpose of this study was to determine the systemic hemodynamic mechanism(s) underlying the pressor response to nonexertional heat stress in the unrestrained conscious rat. After a 60-min control period [ambient temperature (Ta) 24 degrees C], male Sprague-Dawley rats (260-340 g) were exposed to a Ta of 42 degrees C until a colonic temperature (Tc) of 41 degrees C was attained. As Tc rose from control levels (38.1 +/- 0.1 degrees C) to 41 degrees C, mean arterial blood pressure (carotid artery catheter, n = 33) increased from 124 +/- 2 to 151 +/- 2 mmHg (P less than 0.05). During this period, heart rate increased (395 +/- 5 to 430 +/- 6 beats/min, P less than 0.05) and stroke volume remained unchanged. As a result, ascending aorta blood flow velocity (Doppler flow probe, n = 8), used as an index of cardiac output, did not change from control levels during heating, but there was a progressive Tc-dependent increase in systemic vascular resistance (+30% at end heating, P less than 0.05). This systemic vasoconstrictor response was associated with decreases in blood flow (-31 +/- 9 and -21 +/- 5%) and increases in vascular resistance (94 +/- 16 and 53 +/- 8%; all P less than 0.05) in the superior mesenteric and renal arteries (n = 8 each) and increases in plasma norepinephrine (303 +/- 37 to 1,237 +/- 262 pg/ml) and epinephrine (148 +/- 28 to 708 +/- 145 pg/ml) concentrations (n = 12, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of altered ambient temperature on metabolic rate during CO2 inhalation

The purpose of this study was to determine if the changes in O2 consumption (VO2) during CO2 inhalation could in part be due to stimulation of thermogenesis for homeothermy. Twelve ponies were exposed for 30-min periods to inspired CO2 (PIco2) levels of less than 0.7, 14, 28, and 42 Torr during the winter at 5 (neutral) and 23 degrees C ambient temperatures (TA) and during the summer at 21 (neutral TA), 30, and 12 degrees C. Elevating TA in both seasons resulted in an increased pulmonary ventilation (VE) and breathing frequency (f) (P less than 0.01) but no significant increase in VO2 (P greater than 0.05). Decreasing TA in the summer resulted in a decrease in VE and f (P less than 0.01) but no significant change in VO2 (P greater than 0.05). At neutral TA in both seasons, VO2 increased progressively (P less than 0.05) as PIco2 was increased from 14 to 28 and 42 Torr. The increases in VO2 during CO2 inhalation were attenuated (P less than 0.05) at elevated TA and accentuated at the relatively cold TA in the summer (P less than 0.05). Respiratory heat loss (RHL) during CO2 inhalation was inversely related to TA. Above a threshold RHL of 2 cal X min-1 X m-2, metabolic heat production (MHP) increased 0.3 cal X min-1 X m-2 for each unit increase in RHL during CO2 inhalation at the neutral and elevated TA. However, during cold stress in the summer, the slope of the MHP-RHL relationship was 1.6, indicating an increased MHP response to RHL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of temperature and salinity on Vibrio cholerae growth.

Laboratory microecosystems (microcosms) prepared with a chemically defined sea salt solution were used to study effects of selected environmental parameters on growth and activity of Vibrio cholerae. Growth responses under simulated estuarine conditions of 10 strains of V. cholerae, including clinical and environmental isolates as well as serovars O1 and non-O1, were compared, and all strains yielded populations of approximately the same final size. Effects of salinity and temperature on extended survival of V. cholerae demonstrated that, at an estuarine salinity (25%) and a temperature of 10 degrees C, V. cholerae survived (i.e., was culturable) for less than 4 days. Salinity was also found to influence activity, as measured by uptake of 14C-amino acids. Studies on the effect of selected ions on growth and activity of V. cholerae demonstrated that Na+ was required for growth. The results of this study further support the status of V. cholerae as an estuarine bacterium.     

Ultrastructure of coccoid viable but non-culturable Vibrio cholerae

Morphology of viable but non-culturable Vibrio cholerae was monitored for 2 years by scanning and transmission electron microscopy. Morphological changes included very small coccoid forms, after extended incubation at 4 degrees C and room temperature, and sequential transformation from curved rods to irregular (approximately 1 microm) rods to approximately 0.8 microm coccoid cells and, ultimately, to tiny coccoid forms (0.07-0.4 microm). Irregular rod-shaped and coccoid cells were equally distributed in microcosms during the first 30-60 days of incubation at both temperatures, but only coccoid cells were observed after incubation for 60 days at 4 degrees C. When V. cholerae O1 and O139, maintained for 30-60 days at both temperatures, were heated to 45 degrees C for 60 s, after serial passage through 0.45 microm and 0.1 microm filters, and plating on Luria-Bertania (LB) agar, only cells larger than 1 microm yielded colonies on LB agar. Approximately 0.1% of heat-treated cultures were culturable. Cell division in the smallest coccoid cells was observed, yielding daughter cells of equal size, whereas other coccoid cells revealed bleb-like, cell wall evagination, followed by transfer of nuclear material. Coccoid cells of V. cholerae O1 and O139 incubated at 4 degrees C for more than 1 year remained substrate responsive and antigenic.     

Effects of temperature and salinity on Vibrio cholerae growth

Laboratory microecosystems (microcosms) prepared with a chemically defined sea salt solution were used to study effects of selected environmental parameters on growth and activity of Vibrio cholerae. Growth responses under simulated estuarine conditions of 10 strains of V. cholerae, including clinical and environmental isolates as well as serovars O1 and non-O1, were compared, and all strains yielded populations of approximately the same final size. Effects of salinity and temperature on extended survival of V. cholerae demonstrated that, at an estuarine salinity (25%) and a temperature of 10 degrees C, V. cholerae survived (i.e., was culturable) for less than 4 days. Salinity was also found to influence activity, as measured by uptake of 14C-amino acids. Studies on the effect of selected ions on growth and activity of V. cholerae demonstrated that Na+ was required for growth. The results of this study further support the status of V. cholerae as an estuarine bacterium.     

Thermostable, alkalophilic and cellulase free xylanase production by Thermoactinomyces thalophilus subgroup C

Thermoactinomyces thalophilus produced cellulase free extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) at 50 degrees C and pH 8.5. Maximum xylanase production was achieved in fermentation medium using birchwood xylan as substrate after 96 h of growth at 50 degrees C. Other agricultural substrates such as wheat bran, wheat straw, sugarcane bagasse and cornstover produced less xylanase. The crude enzyme preparation from mutant T. thalophilus P2 grown under optimised fermentation conditions showed no cellulase contamination and maximum xylanase activity of 42 U/ml at 65%deg;C and pH 8.5-9.0. This enzyme with initial xylanase activity of 42 U/ml was found thermostable up to 65 degrees C and retaining 50% of its activity after its incubation for 125 min at 65 degrees C.     

Isolation and Characterization of the Fertility Factor P of Vibrio cholerae

Vibrio cholerae strains with the transmissible fertility factor P contained a supercoiled circular deoxyribonucleic acid (DNA) component amounting to between 2 and 6% of the total DNA obtained from the cells. Such a component was not observed in V. cholerae strains lacking the fertility factor. This supercoiled circular DNA was isolated from P(+) cells, and the molecular weight was determined by sedimentation velocity experiments and electron microscopy to be approximately 80 million daltons. These supercoiled circular DNA molecules, which have a guanine plus cytosine (G + C) composition of 42%, were concluded to be the extrachromosomal P factor. It was calculated that there is approximately one copy of the P factor per chromosome. A small amount of supercoiled circular DNA was occasionally isolated from the P(-) strains of V. cholerae. The function of this component, which has a molecular weight of 40 million daltons, is not known. The molecules found in the P(-) strains were readily distinguished from the P(+) circular molecules by their smaller molecular weight and different G + C composition.     

Comparative stability and catalytic and chemical properties of the sulfate-activating enzymes from Penicillium chrysogenum (mesophile) and Penicillium duponti (thermophile).

ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000. (The P. duponti subunit appeared to be a little smaller than the P. chrysogenum subunit.) The P. duponti enzyme was about 100 times more heat stable than the P. chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P. duponti and 3.0 X 10(-2) s-1 for P. chrysogenum. The P. duponti enzyme was also more stable to low pH and urea at 30 degrees C. Rabbit serum antibodies to each enzyme showed heterologous cross-reaction. Amino acid analyses disclosed no major compositional differences between the two enzymes. The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C. At 30 degrees C, the physiologically important adenosine 5'-phosphosulfate (APS) synthesis activity of the P. duponti enzyme was 4 U mg of protein-1, which is about half that of the P. chrysogenum enzyme. The molybdolysis and ATP synthesis activities of the P. duponti enzyme at 30 degrees C were similar to those of the P. chrysogenum enzyme. At 50 degrees C, the APS synthesis activity of the P. duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P. chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1). Treatment of the P. chrysogenum enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit. Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced. Chemical modification with tetranitromethane had the same results on the kinetics. The native P. duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly modified one SH group per subunit. APS kinase (the second sulfate-activating enzyme) of P. chrysogenum dissociated into inactive subunits at 42 degrees C. The P. duponti enzyme remained intact and active at 42 degrees C.