Effects of Host Cell Density on Cell Infection Level in Antheraea eucalypti (Lepidoptera: Saturniidae) Cell Cultures Persistently Infected with Nosema bombycis (Microsporida: Nosematidae)

ABSTRACT. Spores of Nosema bombycis Y9101, isolated from the beet armyworm, Spodoptera exigua , were primed with an alkaline solution and inoculated into Antheraea eucalypti cell cultures. Infected cells were subcultured every five days at three cell densities (2.5 × 10³, 5.0 × 10³, and 1.0 × 10⁴ cells/cm²). A difference was observed in the spread of N. bombycis Y9101 infection between low-density and higher-density cultures of host cells. The host cell density did not affect the productivity of secondary infective forms of the parasite. The principal factor determining the rate of microsporidian infection in vitro was the number of host cells existing within the reach of extruded short-coiled polar tubes from spores germinated intracellularly.     

Isolation of an inclusion body from sporulating, enterotoxin-positive Clostridium perfringens

Abstract Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens were isolated. Sporulating cells were lysed by sonication in the presence of protease inhibitors. IB were isolated by centrifugation in linear gradients of sucrose, sodium bromide or sodium diatrizoate and banded at buoyant densities of 1.33–1.36 g/cm³, 1.30–1.34 g/cm³ and 1.33 g/cm³, respectively. Isolated IB were treated with detergent to remove attached cell membrane. They ranged in size from 0.5–1.4 μm long and from 0.2–0.5 μm wide. They were found to be serologically related to purified enterotoxin.     

Cell proliferation and polyamine metabolism in 9L cells treated with (2R,5R)-6-heptyne-2,5-diamine or α-difluoromethylornithine

Abstract We studied the effects of the ornithine decarboxylase inhibitors (2R,5R)-6-heptyne-2,5-diamine (R,R,-MAP) and α-difluoromethylornithine (DFMO) on cell proliferation and polyamine metabolism in 9L rat brain tumour cells. Treatment with 5 μM R,R-MAP inhibited cell proliferation to the same extent as did treatment with 1 mM DFMO. Both inhibitors depleted putrescine and spermidine concentrations to less than detectable levels within 24 h and 48 h of drug treatment, respectively; spermine levels were not affected significantly by either inhibitor. The effects of DFMO on 9L cell cycle kinetics were similar to those of R,R-MAP. During the first 3 days of treatment, both drugs caused an accumulation of cells in G₁ and a reduction of cells in S phase, as compared with control cells with a slowing in the rate of cell cycle traverse. In cultures seeded at low (1 × 10⁵), medium (5 × 10⁵), or high (2 × 10⁶) cell densities in a 25 cm² flask, inhibition of cell proliferation and polyamine depletion by both R,R-MAP and DFMO was more pronounced at the lower densities relative to the density-matched control cells. Thus, R,R-MAP was a more potent inhibitor of ornithine decarboxylase than was DFMO in 9L cells, and the inhibitory effects of both compounds on cell proliferation and polyamine biosynthesis were greater in actively proliferating cells.     

Changes of Nucleic Acids and Proteins in the Oocysts of Eimeria tenella during Sporulation

SYNOPSIS. The total content of DNA in Eimeria tenella , estimated at 5.8 × 10⁻¹² gm/oocyst, varies little during sporulation. Its buoyant density is 1.682 gm/cm³, reflecting a G + C content of ∼41%. Thymidine is not incorporated into any TCA insoluble fraction of sporulating oocysts, but radioactivity from [³H]uridine and [³H]deoxyuridine are incorporated into RNA at a linear rate during the first 5 hr of sporulation. The labeled RNA, found mainly in the paranuclear bodies of newly formed sporozoites, contains ∼0.15 nmole [³H]uridine/10⁶ oocysts at the completion of sporulation. One nmole of leucine is incorporated into the hot TCA insoluble fraction of 10⁶ oocysts during the first 7 hr of sporulation after an initial lag. The incorporated amino acid is mainly in the cytoplasm of the sporozoites, and an analysis by SDS-gel electrophoresis reveals most of the radioactivity in a narrow band with a molecular weight of ∼50,000 daltons. Incorporation of uridine and leucine, however, can be totally suppressed by respiratory inhibition. Further analysis of the proteins in the oocysts reveals that the total protein content remains relatively unchanged at 2.64 × 10⁻¹⁶ gm/oocyst during sporulation, but there is a shift of 13–14% of total protein from the soluble cytoplasm to the 15,000 g pellets. By polyacrylamide gel electrophoresis, a major protein band. possibly a glycoprotein, is shown in the soluble cytoplasm of unsporulated oocysts. This band disappears during sporulation.     

EFFECT OF DISSOLVED OXYGEN PARTIAL PRESSURE ON THE ACCUMULATION OF ASTAXANTHIN IN CHEMOSTAT CULTURES OF HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)

The effect of dissolved oxygen partial pressure on the accumulation of astaxanthin in the green alga Haematococcus lacustris ( Gir.) Rostaf (UTEX16) was studied in N-limited continuous chemostat cultures. The steady-state astaxanthin content measured against culture volume, cell number, and biomass dry weigh of Haematococcus cultures was proportional to the dissolved O₂ partial pressure in the culture medium, over the range of 0–50% O₂ The steady-state biomass dry weight concentrations remained at between 0.52 and 0.57 g. L^-1 over the range of dissolved O₂ partial pressure studied. Steady-state cell densities at dissolved O₂ partial pressures above the air saturation level (1.13–1.58 × 10⁵ cells.mL^-1) were about half of that measured at lower dissolved O₂ partial pressures (2.42–2.63 × 10⁵ cells.mL^-1). Both biflagellated zoospores and nonmotile aplanospores were found at steady state. The fraction of nonmotile cells was higher at dissolved O₂ partial pressures above the air saturation level (94.44–98.01%) than at dissolved O₂ partial pressure below the air level (79.64–86.12 and 91.75% ).     

EVALUATION OF ADENOSINE TRIPHOSPHATE (ATP) BIOLUMINESCENCE FOR ESTIMATING BACTERIA ON SURFACES OF BEEF CARCASSES

A rapid (<15 min), inexpensive and simple method has been developed to estimate the concentration of bacteria on surfaces of beef carcasses using adenosine triphosphate (ATP) bioluminescence. Surfaces (5x5 cm²) of beef carcasses (n= 159) were collected by excision. An ATP assay and aerobic plate count were performed on each sample. A significant (p < 0.001) positive linear relationship (r = 0.83) between plate count and ATP assay was obtained for 159 beef carcass samples. When thresholds levels were set at 1 × 10⁴, 1 × 10⁵ and 1 × 10⁶ CFU/cm², there was moderate to good agreement between the ATP bioluminescence assay and the aerobic plate count as determined by the k-statistic. The application of this ATP bioluminescence test to HACCP systems for beef slaughter processes is discussed.     

A note on the use of 3-section plates for the estimation of the numbers of bacteria and to obtain isolated colonies in 1 day

By streaking with an open loop and then swabbing a 4–5 cm² area on 3-section agar plates, it is possible to obtain isolated colonies and to estimate bacterial densities from 100 to 10⁷/ml on the swabbed area.     

Generation Time and Growth Rate of the Human Intestinal Parasite Blastocystis hominis

ABSTRACT. Blastocystis hominis , an anaerobic intestinal protozoan parasite of man, has a generation time (GT) in axenic culture of 8.5–19.4 h, depending on the strain tested. Average GT of the eight strains was 11.7 h. Zero growth time cell counts of 5.0 × 10⁵/ml to 2.0 × 10⁶/ml rose in 3–5 days to 1 × 10⁷ or 1 × 10⁸ cells/ml. The GT was determined for the 24-h period during which the most rapid growth occurred; about 2% of the B. hominis cells were in division during this time. Division under the culture conditions provided was by binary fission, the usual mode for B. hominis in vitro as well as in vivo. Division times were determined also by direct observation of individual dividing cells in slide cultures. These were usually ca. 40–60 min but sometimes as low as 20 min.     

CELL SURFACE PROTEINS OF CULTURED BRAIN CELLS AND THEIR RECOGNITION BY ANTI-CEREBELLUM (ANTI-NS-4) ANTISERUM

Abstract— Surface proteins of cultured young postnatal mouse cerebella and embryonic mouse cerebral hemispheres were identified by Iactoperoxidase-catalysed radioiodination and by their interaction with an anti-mouse cerebellum antiserum (anti-NS-4 serum) which recognizes surface components on brain cells. Several (8 10) iodinated polypeptides are recognized by radioautography after polyacrylamide gel electrophoresis. Their surface location was confirmed by their sensitivity to mild trypsin treatment on intact cells. Iodinated polypeptides from cells of non-nervous tissues showed a different gel pattern. Immuno-precipitates of solubilizcd surface-iodinated cerebellar cells with anti-NS-4 serum contained two prominent labeled proteins with apparent molecular weights of 200 × 10³ and 145 × 10³. These proteins were also biosynthetically labeled with [³H]leucine. The 145 × 10³ molecular weight component was also found in immunoprecipitates prepared from embryonic cerebral cells, but the 200 × 10³ molecular weight component was replaced by a broad peak with an apparent molecular weight of around 250 × 10³.     

Spontaneous All-or-Nothing Action Potentials in the Ciliate Bursaridium difficile

The electrical membrane properties and the swimming behaviour of the freshwater ciliate Bursaridium difficile were studied by current clamp recordings and video analysis. The resting membrane potential was –45 ± 6 mV (mean ± SD, n = 80), and the input resistance and membrane capacitance were 109 ± 42 megaohms (MΩ) (n = 63) and 457 ± 150 picofarads (pF) (n = 42), respectively. Based on an estimated surface area of 6.8 × 10^-4 cm², the corresponding specific membrane resistance and capacitance are 7.4 × 10⁴Ω× cm² and 0.7 μF/cm². Bursaridium difficile generates spontaneous, all-or-nothing action potentials with a well-defined threshold in normal medium. The spontaneous firing frequency was 0.22 ± 0.06 Hz (n = 80). The maximum rate of rise of the action potentials was less than 1 V/s, and they displayed a prolonged plateau phase (0.5–1 s). The action potentials were abolished in nominal Ca²⁺-free solution and are thus Ca²⁺-spikes. The swimming pattern of Bursaridium in homogeneous surroundings is composed of forward swimming periods interrupted by regular, short periods of backward swimming followed by a change in the forward swimming direction. The turning frequency corresponded to the spontaneous firing frequency, and only forward swimming was observed in nominal Ca²⁺-free solution. The periods of backward swimming activity are thus linked to the spontaneous action potentials.     

Experimental Infection of Nosema sp. Ghose in an Unnatural Host Lesioderma sericorne

ABSTRACT. Various doses of a microsporan parasite, Nosema sp., were fed to third and fourth instar larvae of Lesioderma sericorne that infested different types of stored grains. A spore dose of 3 × 10³ spores/individual resulted in a 39% infection rate, reduction in larval and adult weights, and mean spore concentrations of 1.28 ± 0.2 × 10⁸ spores/larva and 1.1 ± 0.2 × 10⁸ spores/adult. At the above dose, mortality was not well marked (about 35% in larvae and 25% in adults). At 3 × 10⁴ spores/individual, the rate of mortality increases to 80% in larvae and 60% in adults. However, more of the pest population (88% of larvae and 73% of adults) died at a dose of 3 × 10⁵ spores/individual. This dose produced mean spore concentrations of 3.91 ± 0.2 × 10⁸ spores/larva and 2.89 ± 0.2 × 10⁸ spores/adult. Insect death was caused by heavy damage to gut epithelia and fat bodies.     

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.     

Rates of sulfate reduction and thiosulfate consumption in a marine microbial mat

Abstract The sulfur cycle in a microbial mat was studied by determining viable counts of sulfate-reducing bacteria, chemolithoautotrophic sulfur bacteria and anoxygenic phototrophic bacteria. All three functional groups of sulfur bacteria revealed a maximum population density in the uppermost 5 mm of the mat: 1.1 × 10⁸ cells of sulfate reducers cm⁻³ sediment, 2.0 × 10⁹ cells of chemolithoautotrophs cm⁻³ sediment, and 4.0 × 10⁷ cells of anoxygenic phototrophs cm⁻³ sediment. Bacterial dynamics were studied by sulfate reduction rate measurements, both under anoxic conditions (dark incubation) and oxic conditions (incubation in the light), and determination of the vertical distribution of the potential rate of thiosulfate consumption under oxic conditions. Sulfate reduction rates in the top 5 mm of the sediment were 566 nmol cm⁻³ d⁻¹ in the absence of oxygen, and 123 nmol cm⁻³ d⁻¹ in the presence of oxygen. In the latter case, the maximum rate was found in the 5–10-mm depth horizon (361 nmol cm⁻³ d⁻¹). Biological consumption of amended thiosulfate was rapid and decreased with depth, while in the presence of molybdate, thiosulfate consumption decreased to 10–30% of the original rate.     

Source of cell injected is a critical factors for short and long engraftment in xeno-transplantation

Abstract. This study aims to investigate engraftment of human cord blood and foetal bone marrow stem cells after in utero transplantation via the intracoelomic route in the sheep. Here, we performed transplantation in 14 single and 1 twin sheep foetuses at 40–47 days of development, using a novel schedule for injection. (i) Single injection of CD34⁺ human cord blood stem cells via the coelomic route (from 10 to 50 × 10⁴) in seven single foetuses. (ii) Single injection of CD34⁺ foetal bone marrow stem cells via the intracoelomic route with further numbers of cells (20 × 10⁵ and 8 × 10⁵, respectively) in three single and in one twin foetuses. (iii) Double fractioned injection (20–30 × 10⁶) via the coelomic route and 20 × 10⁶ postnatally, intravenously, shortly after birth of CD3-depleted cord blood stem cells in four single foetuses. In the first group, three single foetuses showed human/sheep chimaerism at 1, 8 and 14 months after birth. In the second group, the twin foetuses showed human/sheep chimaerism at 1 month after birth. In the third group, only two out of four single foetuses that underwent transplantation showed chimaerism at 1 month. While foetal bone marrow stem cells showed good short-term engraftment (1 month after birth), cord blood stem cells were able to persist longer in the ovine recipients (at 1, 8 and 14 months after birth).     

A note on the control of sulphate-reducing bacteria in seawater by u.v. irradiation

Continuous and batch cultures of marine sulphate-reducing bacteria (SRB) in North Sea water were irradiated with 110000 to 329500 μWs/cm² of ultraviolet radiation (wavelength 253.7 nm) with a commercial u.v. sterilizing unit. A 100% kill was obtained with logarithmic cultures of Desulfovibrio desulfuricans NCIMB 8400 at population densities of 10–10⁴/ml. A >99.99% kill was obtained with a mixture (ca 10⁵/ml) of batch grown Desulfovibrio spp. and oilfield SRB enrichments. Ultraviolet irradiation was less effective against the indigenous heterotrophic bacteria in the seawater ( ca 90% kill).     

Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon, Salmo salar

Blood samples from healthy adult Atlantic salmon fed an optimal diet in net sea pens were collected at intervals from October to May. Haematological determinations and biochemical serum analyses were carried out on 20 fish in each of seven samples. The ranges of haemato-logical values for sample means were: haematocrit 44–49%, haemoglobin 8.9–10.4 g 100ml⁻¹, red blood cell count 0.85–1.10 × 10¹² l⁻¹, MCV 441–553 × 10⁻¹⁵ 1, MCH 94–106 × 10⁻⁶ g, MCHC 19.4–21.7 g 100ml⁻¹ and leucocrit 0.43–0.96%. The ranges of enzyme activities in serum, for sample means, were: alkaline phosphatase 647–988Ul⁻¹, aspartate aminotrans-ferase 202–351 Ul⁻¹ and alanine aminotransferase 4–8 Ul⁻¹. The ranges of the other parameters analyzed in serum were: total protein 41.6–56.6 gl⁻¹, albumin 18.3–24.3 gl⁻¹, albumin/total protein ratio 39.3–44.0%, creatinine 26–46 μmol, triglycerides 2.53–4.98 μmol and cholesterol 9.3–12.8 μmol. These values are considered to be the normal ranges in healthy fish. Variations due to seasonal changes, and the clinical significance of the selected parameters, are discussed. Data showing the reproducibility of the biochemical analyses in serum are presented.     

Competition between anoxygenic phototrophic bacteria and colorless sulfur bacteria in a microbial mat

Abstract The populations of chemolithoautotrophic (colorless) sulfur bacteria and anoxygenic phototrophic bacteria were enumerated in a marine microbial mat. The highest population densities were found in the 0–5 mm layer of the mat: 2.0 × 10⁹ cells cm⁻³ sediment, and 4.0 × 10⁷ cells cm⁻³ sediment for the colorless sulfur bacteria and phototrophs, respectively. Kinetic parameters for thiosulfate-limited growth were assessed for Thiobacillus thioparus T5 and Thiocapsa roseopersicina M1, both isolated from microbial mats. For Thiobacillus T5, growing at a constant oxygen concentration of 43 μmol l⁻¹, μ_max was 0.336 h⁻¹ and K _s 0.8 μmol l⁻¹. Phototrophically grown Thiocapsa strain M1 displayed a μ_max of 0.080 h⁻¹ and a K _s of 8 μmol l⁻¹ when anoxically grown under thiosulfate limitation. In a competition experiment with thiosulfate as electron donor, Thiocapsa became dominant during a 10-h oxic/14-h anoxic regimen at continuous illumination, despite the higher affinity for thiosulfate of Thiobacillus .     

Multiplication of Pseudomonas syringae pv. phaseolicola "in planta"

Multiplication of Pseudomonas phaseolicola was determined in 17 different bean cultivars ( Phaseolus vulgaris ) and 9 other plant species, and the effect of different inoculation methods and conditions was also studied.
In susceptible leaves, a generation time of 2.1 h was determined in the early phase (2 days after inoculation). Different multiplication rates in susceptible and resistant leaves were clearly observed 4 days after inoculation. At this time the first small water-soaked spots were visible in the susceptible cultivars. Bacteria multiplied up to the 7th day after inoculation with a maximum of 10⁹ cells per cm² leaf (equal to ca. 4 × 10¹⁰ bacterial cells/cm³). At the same time, the water-soaked spots had reached their maximum size in most cases. Thus, bacterial multiplication and development of water-soaked spots paralleled each other.
In resistant leaves, no water-soaked spots appeared, and the final bacterial concentration was 1/1000–1/100 of that in susceptible leaves. Gomparison of races 1 and 2 in several bean cultivars indicated the non-existence of a gene-for-gene relationship with this disease. Old leaves were less susceptible to infection. Some bacterial multiplication was also observed in non-host plants. There was a general correlation between bacterial multiplication in the non-host plants and their botanical relation to Phaseolus vulgaris .     

Isolation of Pectolytic clostridia from Potatoes

S ummary : A method for selective counting and isolation of pectolytic clostridia in the presence of Erwinia carotovora is described; using this method pectolytic clostridia have been found in numbers of 8 × 10⁵–1 × 10⁸/g of rotting potato tissue in the presence of 1–4 × 10⁸ E. carotovora /g.     

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.