Antibacterial Activity Evaluation of Microcin J25 against Diarrheagenic Escherichia coli

The inhibitory activities of known microcins were evaluated against some diarrheagenic Escherichia coli strains. Some antibacterial properties of microcin J25, the most active one, were studied. A rapid two-step purification was performed. The MIC and the minimum bactericidal concentration of J25 against E. coli O157:H7 were 1 and 100 μg ml⁻¹, respectively. A 10⁴-CFU ml⁻¹ contamination by this strain was destroyed in milk and meat extract by 6.25 μg of J25 ml⁻¹ and in half-diluted egg yolk by 50 μg of J25 ml⁻¹.     

Chloroform-Methanol Residue of Coxiella burnetii Markedly Potentiated the Specific Immunoprotection Elicited by a Recombinant Protein Fragment rOmpB-4 Derived from Outer Membrane Protein B of Rickettsia rickettsii in C3H/HeN Mice

The obligate intracellular bacteria, Rickettsia rickettsii and Coxiella burnetii, are the potential agents of bio-warfare/bio-terrorism. Here C3H/HeN mice were immunized with a recombinant protein fragment rOmp-4 derived from outer membrane protein B, a major protective antigen of R. rickettsii, combined with chloroform-methanol residue (CMR) extracted from phase I C. burnetii organisms, a safer Q fever vaccine. These immunized mice had significantly higher levels of IgG1 and IgG2a to rOmpB-4 and interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), two crucial cytokines in resisting intracellular bacterial infection, as well as significantly lower rickettsial loads and slighter pathological lesions in organs after challenge with R. rickettsii, compared with mice immunized with rOmpB-4 or CMR alone. Additionally, after challenge with C. burnetii, the coxiella loads in the organs of these mice were significantly lower than those of mice immunized with rOmpB-4 alone. Our results prove that CMR could markedly potentiate enhance the rOmpB-4-specific immunoprotection by promoting specific and non-specific immunoresponses and the immunization with the protective antigen of R. rickettsii combined with CMR of C. burnetii could confer effective protection against infection of R. rickettsii or C. burnetii.     

Effects of Aldicarb and Its Sulfoxide and Sulfone on the Biology of Tylenchulus semipenetrans

In laboratory testing, egg hatch of Tylenchulus semipenetrans was stimulated at concentrations of 1 and 10 μg/ml aldicarb solution and inhibited at 50 and 100 μg/ml. Aldicarb was more inhibitory to egg hatch than the aldicarb sulfoxide and the aldicarb sulfone. Inhibition of hatch at the high concentration was associated with delays in the molting processes, lack of larval movement within the egg, and delays in embryonic development. Nematode motility was reduced at 10, 50, and 100 μg/ml of aldicarb and aldicarb sulfoxide solution, and at 50 and 100 μg/ml aldicarb sulfone. Male development was retarded at 10 μg/nrl and almost completely inhibited at 50 and 100 μg/ml of the three chemicals. In greenhouse tests, female development antl reproduction on roots of citrus seedlings were suppressed by aldicarb at rates of 2.6 μg/ml and completely inhibited at 10.6 μg/ml of soil solution during a 50-day experimental period. Under field conditions, there was little systemic movement of aldicarb into roots located outside treated areas. Aldicarb reduced the nematode larvae and the female adult population in the second year after the second treatment. There were no differences in egg hatch and sex ratio of citrus nematodes between treated and nontreated roots.     

Effect of Dietary Vitamin A on Reproductive Performance and Immune Response of Broiler Breeders

The effects of dietary vitamin A supplementation on reproductive performance, liver function, fat-soluble vitamin retention, and immune response were studied in laying broiler breeders. In the first phase of the experiment, 1,120 Ross-308 broiler breeder hens were fed a diet of corn and soybean meal supplemented with 5,000 to 35,000 IU/kg vitamin A (retinyl acetate) for 20 weeks. In the second phase, 384 Ross-308 broiler breeder hens were fed the same diet supplemented with 5,000 to 135,000 IU/kg vitamin A (retinyl acetate) for 24 weeks. The hens'' reproductive performance, the concentrations of vitamins A and E in liver and egg yolk, liver function, mRNA expression of vitamin D receptor in duodenal mucosa, antibody titers against Newcastle disease virus vaccine, and T-cell proliferation responses were evaluated. Supplementation of vitamin A at levels up to and including 35,000 IU/kg did not affect reproductive performance and quadratically affected antibody titer to Newcastle disease virus vaccine (p<0.05). Dietary addition of vitamin A linearly increased vitamin A concentration in liver and yolk and linearly decreased α-, γ-, and total tocopherol concentration in yolk (p<0.01) and α-tocopherol in liver (p<0.05). Supplementation of vitamin A at doses of 45,000 IU/kg and above significantly decreased egg weight, yolk color, eggshell thickness and strength, and reproductive performance. Dietary vitamin A significantly increased mRNA expression of vitamin D receptor in duodenal mucosa (p<0.05), increased aspartate amino transferase activity, and decreased total bilirubin concentration in serum. Supplementation of vitamin A at 135,000 IU/kg decreased the proliferation of peripheral blood lymphocytes (p<0.05). Therefore, the maximum tolerable dose of vitamin A for broiler breeders appears to be 35,000 IU/kg, as excessive supplementation has been shown to impair liver function, reproductive performance, and immune response.     

BIOCHEMICAL CYTOLOGY OF TRICHOMONAD FLAGELLATES : I. Subcellular Localization of Hydrolases, Dehydrogenases, and Catalase in Tritrichomonas foetus

To determine the localization of several enzymes in Tritrichomonas foetus, the axenic KV-1 strain was grown in Diamond's medium with bovine serum, homogenized in 0.25 M sucrose, and subjected to analytical differential and isopycnic centrifugation. The fractions were assayed for their enzymatic composition and examined electron microscopically. NADH and NADPH dehydrogenases, about 90% of the catalase, and two hydrolases, α-galactosidase and manganese-activated β-galactosidase I are in the nonsedimentable part of the cytoplasm. α-Glycerophosphate and malate dehydrogenases are associated with a large particle, whose equilibrium density in sucrose gradients is 1.24. This particle corresponds to that population of the paracostal and paraxostylar granules which, having a uniform granular matrix surrounded by a single membrane, resemble microbodies from other organisms. The small sedimentable portion of catalase (about 10% of the total activity) is not associated with these granules and equilibrates at density 1.22. The nature of the subcellular entity carrying catalase could not be ascertained. Hydrolases with a pH optimum around 6–6.5 (protease, β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase, and cation-independent β-galactosidase II), as well as a large part of acid phosphatase, are associated with a population of large particles which equilibrate at densities from 1.15 to 1.20. The hydrolases in these granules lose their structure-bound latency easily after freezing and thawing. These particles correspond to another population of the paracostal and paraxostylar granules which have varied shape and inhomogeneous content with frequent myelin figures, indicating a digestive function. The rest of the phosphatase and most of the acid β-glucuronidase activity are in a smaller granule fraction with an equilibrium density around 1.18. The latency of these enzymes is quite resistant to freezing and thawing. This particle population consists of smaller, very often flattened vesicles and granules, many of which are clearly fragments of the prominent Golgi apparatus of the cell.     

Effects of Host Resistance on the Fecundity of Globodera rostochiensis

The fecundity of Globodera rostochiensis (R₁A) females that developed on resistant Rosa and susceptible Katahdin potato cultivars were compared. Cysts collected from each cultivar were bulked, separated into four sizes (> 500 μm, 355-500 μm, 250-355 μm, and < 250 μm), and crushed to determine fecundity as measured by viable egg content (VEC). Fewer and generally smaller cysts developed on Rosa than on Katahdin. Although cyst size significantly (P = 0.01) influenced VEC, cyst age (8 or 13 weeks) had no effect. Regardless of size, cysts produced on Rosa contained significantly fewer viable eggs than did cysts produced on Katahdin. The fecundity of progeny from cysts produced on Rosa was significantly reduced compared with that of progeny from cysts produced on Katahdin. After two generations on Katahdin, the VEC of cysts from a population originating from Rosa was significantly less than that of cysts from a population originating from Katahdin, indicating that in the presence of a pure population of G. rostochiensis R₁A, the females that develop on the resistant cultivar Rosa represent a diminished rather than a superior selected population.     

Population Size and Distribution of Rhizobium leguminosarum bv. trifolii in Relation to Total Soil Bacteria and Soil Depth

Bacterial cells small enough to pass through 0.4-μm-pore-size filters made up 5 to 9% of the indigenous bacterial population in 0- to 20-cm-depth samples of Abiqua silty clay loam. Within the same soil samples, cells of a similar dimension were stained with fluorescent antibodies specific to each of four antigenically distinct indigenous serogroups of Rhizobium leguminosarum bv. trifolii and made up 22 to 34% of the soil population of the four serogroups. Despite the extensive contribution of small cells to these soil populations, no evidence of their being capable of either growth or nodulation was obtained. The density of soil bacteria which could be cultured ranged between 0.5 and 8.5% of the >0.4-μm direct count regardless of media, season of sampling, or soil depth. In the same soil samples, the viable nodulating populations of biovar trifolii determined by the plant infection soil dilution technique ranged between 1 and 10% of the >0.4-μm direct-immunofluorescence count of biovar trifolii. The <0.4-μm cell populations of both total soil bacteria and biovar trifolii changed abruptly between the 10- to 15-cm and 15- to 20-cm soil depth increments, increasing from 5 to 20% and from 20 to 50%, respectively, of their direct-count totals. The increase in density of the small-cell population corresponded to a significant increase in soil bulk density (1.07 to 1.21 g cm⁻³). The percent contribution of the <0.4-μm direct count to individual serogroup totals increased with soil depth by approximately 2-fold (39 to 87%) for serogroups 17 and 21 and by 12-fold (6 to 75%) for serogroups 6 and 36.     

Practical Method for Extraction of PCR-Quality DNA from Environmental Soil Samples

Methods for the extraction of PCR-quality DNA from environmental soil samples by using pairs of commercially available kits were evaluated. Coxiella burnetii DNA was detected in spiked soil samples at <1,000 genome equivalents per gram of soil and in 12 (16.4%) of 73 environmental soil samples.The detection of pathogenic organisms in the environment often relies on PCR analysis of DNA purified from environmental soil (6). For effective detection, a reliable method to obtain PCR-quality DNA from soil is necessary. Although a variety of complex techniques have been effective for specific soil samples (1-3, 7, 8), it is not clear which methods would be the best for the wide variety of samples encountered in a large-scale environmental sampling study. In addition, many published techniques would be difficult to use on a large number of samples (1-3, 7, 8).This study evaluates the abilities of commercially available DNA extraction kits to provide DNA from environmental soil samples that are suitable for PCR detection of Coxiella burnetii. C. burnetii is an obligate intracellular, Gram-negative, zoonotic pathogen and the causative agent of Q fever (5). It is classified as a category B agent of bioterrorism by the CDC.Three commercially available DNA purification kits were evaluated. Twenty different soil samples obtained from diverse locations in the southeastern United States were used for testing. These samples consisted of light sandy soil and were all initially processed through one of three DNA purification kits, the UltraClean soil DNA isolation kit (MoBio Laboratories, Carlsbad CA), the QIAamp DNA minikit (Qiagen, Valencia, CA), or the QIAamp DNA stool minikit (Qiagen), or through a combination of two of the kits used sequentially. Thus, all 20 samples were each processed through nine extraction protocols. To process soil samples, five grams of soil was mixed with 10 to 30 ml of phosphate-buffered saline (PBS) to create a homogenized slurry. Samples were mixed for 1 h at room temperature and then centrifuged for 5 min at 123 × g. The supernatant was removed and centrifuged at 20,000 × g for 15 min. The supernatant was then carefully discarded and the pellet resuspended in 1 ml of PBS.For the UltraClean soil kit, 700 μl of the resuspended soil extraction pellet was processed by the manufacturer''s alternative protocol (for maximum yields). For preps done using the QIAamp DNA minikit (tissue protocol) and the QIAamp stool kit (stool protocol), 700 μl (high volume) of the soil extract was processed according to the instructions for the particular kit. For 17 of the samples the tissue protocol and stool protocol were applied using only 200 μl of the soil extract (low volume). For all of the kits, the final elutions were performed with 55 μl of water.To further purify the products of the commercial DNA isolation kits, eluates were passed through a second round of extraction. When the MoBio UltraClean kit was used for the second round of extraction, eluates were added to the bead-containing tubes and mixed with 60 μl of solution 1 and 200 μl of the MoBio inhibitor removal solution (IRS). The manufacturer''s protocol was then followed. When the QIAamp tissue protocol was utilized for the second round of extraction, eluates were diluted to 200 μl with water and then mixed with 200 μl of buffer ATL plus 200 μl of buffer AL and then incubated at 70°C for 10 min. Following this step, the manufacturer''s protocol was followed. When the QIAamp stool protocol was used for the second round of extraction, eluates were mixed with 1.2 ml of the ASL buffer, followed by addition of the InhibitEX tablet. The manufacturer''s protocol was then followed.PCR inhibition in all of the DNA samples was then evaluated by running a quantitative PCR that detects the IS1111 gene from C. burnetii (4). PCRs were run on 200 genome equivalents of C. burnetii (strain Nine Mile Phase 1) DNA. Reaction mixtures spiked with 1-μl aliquots of the environmental DNA samples were compared to reaction mixtures spiked with 1 μl of water. Inhibition was considered present if the DNA sample caused an increase of 1 in the threshold cycle value.Use of the MoBio UltraClean procedure by itself resulted in removal of inhibitors from 35% of the samples, whereas after use of the Qiagen tissue protocol (high volume) only 4% of the samples were free of inhibition (Fig. ​(Fig.1).1). The Qiagen stool kit (high volume) resulted in 96% of the samples showing lack of inhibition with a low volume of soil eluate and 62.5% of the samples when the high volume was used. The DNA extracted from these three kits was then used as starting material for a subsequent DNA extraction step using the same set of three commercial kits. The MoBio UltraClean kit followed by the Qiagen stool kit eliminated inhibition in all samples, as did these two kits when used in the reverse order, even if the Qiagen stool kit was loaded with 700 μl of material (high volume). When a low volume of starting material was used, combinations of the two Qiagen kits also removed inhibitors from 100% of the samples when either the Qiagen tissue protocol was used first or the Qiagen stool protocol was used first (Fig. ​(Fig.1).1). The raw data for all of the inhibition assays are included as supplemental data (see Table S1 in the supplemental material).Open in a separate windowFIG. 1.Twenty environmental soil samples were used for the isolation of DNA with the indicated protocols. The samples were then tested for the ability to inhibit an IS1111 PCR with C. burnetii Nine Mile DNA as template. The percentages of samples that did not show any inhibition are indicated.To determine the yield of DNA obtained by the various protocols, nine aliquots (5 g each) of a single rich organic soil sample were each mixed with 5 ml PBS, spiked with 1 × 10⁶ Nine Mile Phase 2 C. burnetii organisms, and then processed by the nine (high-volume) extraction protocols described above. An additional 1 × 10⁶ Nine Mile Phase 2 C. burnetii organisms were used directly in the Qiagen tissue protocol to prepare DNA for the purpose of determining the exact amount of C. burnetii input into the assays. The quantitative IS1111 PCR assay (4) was used to determine the yield of C. burnetii DNA by using the various methods for processing soil. The yield was calculated by dividing the number of genome equivalents of C. burnetii DNA obtained from the spiked soil samples by the number of genome equivalents obtained when C. burnetii was included directly in the Qiagen tissue protocol. A common feature of all of the protocols was that they all produced a low yield of C. burnetii DNA when purified from a complex soil mixture (Fig. ​(Fig.2).2). The yields ranged from 0.02% to 4.3% and were variable. Although the 4.3% yield obtained when the stool kit was used alone was the highest on average, the high variability observed with these extractions suggests that most of these protocols provide similar yields. The stool kit followed by the MoBio kit clearly resulted in the lowest yield.Open in a separate windowFIG. 2.Five-gram aliquots of a single soil sample were all spiked with approximately 1 × 10⁶ C. burnetii Phase 2 Nine Mile strain cells. The samples were then subjected to the indicated extraction protocol(s). The resulting DNA was tested for inhibition, and then the genome equivalents of C. burnetii DNA were determined by quantitative IS1111 PCR. The exact input amount of C. burnetii was determined by running an aliquot directly through the QIAamp tissue protocol followed by IS1111 PCR. Yield was calculated as genome equivalents obtained from the spiked soil samples divided by the genome equivalents obtained from the direct extraction through the QIAamp tissue protocol. Values represent the mean ± standard deviation of five experiments. Statistically significant differences (Student''s t test) were found between stool versus MoBio plus stool kits (P = 0.05), stool plus tissue versus MoBio plus stool kits (P = 0.01), and stool plus tissue versus tissue plus MoBio kits (P = 0.03). For the protocol using the stool kit followed by the MoBio kit the yield was significantly different from stool, stool plus tissue, MoBio plus tissue, and MoBio protocols (P < 0.05).Although these yields are low, the IS1111 PCR assay used to detect C. burnetii DNA amplifies a multicopy gene, and the assay can detect a single genome equivalent (4). This suggests that these protocols are adequate for the detection of C. burnetii in soil samples with 500 to 2,000 organisms per gram of soil. To test this, a 5-g sample of organic soil was spiked with 800 C. burnetii organisms per gram, and the DNA was extracted using the MoBio UltraClean kit followed by the QIAamp stool protocol. C. burnetii DNA was detected after 38 cycles using the IS1111 PCR assay.While these results are focused on soil samples, the procedures described also work well on vacuum samples and sponge wipe samples (data not shown). Based on removal of inhibitors and yield, our data suggest that the QIAamp tissue protocol (high volume) followed by the QIAamp stool protocol and the MoBio UltraClean kit followed by the QIAamp stool protocol are both suitable for extraction of DNA from environmental soil samples. To test the application of the latter method to a larger number of samples, 73 bulk soil samples from the southeastern United States were processed according to this method. Inhibition was removed from all 73 samples, and 12 of the samples were positive in the C. burnetii IS1111 PCR assay. This suggests that this practical method for extraction of PCR-quality DNA can be successfully used to detect DNA from C. burnetii and other pathogens in large numbers of environmental samples.      

Volatile Fatty Acid Requirements of Cellulolytic Rumen Bacteria

A gas chromatographic method was developed which could separate the isomers isovaleric and 2-methylbutyric acid. Subsequent analyses revealed that most commercially available samples of these acids were cross-contaminated; however, one sample of each acid was found to be pure by this criterion. The growth response of seven strains of cellulolytic rumen bacteria (three strains of Bacteroides succinogenes, three strains of Ruminococcus flavefaciens, and one strain of R. albus) to additions of isobutyric, isovaleric, 2-methylbutyric, valeric, and combinations of valeric and a branched-chain acid was determined. Strains of B. succinogenes required a combination of valeric plus either isobutyric or 2-methylbutyric acid. Isovaleric acid was completely inactive. Either isobutyric or 2-methylbutyric acid was required for the growth of R. albus 7. Strain C-94 of R. flavefaciens grew slowly in the presence of any one of the three branched-chain acids, but a combination of isobutyric and 2-methylbutyric acids appeared to satisfy this organism's growth requirements. None of the individual acids or mixtures of straight- and branched-chain acids allowed growth of R. flavefaciens strain C1a which would approach the response obtained from the total mixture of acids. Further work indicated that all three branched-chain acids were required for optimal growth by this strain, although isovaleric acid only influenced the rate of maximal growth. Either 2-methylbutyric or isovaleric acid allowed growth of nearly the same magnitude as that of the positive control for R. flavefaciens B34b. The presence of acetic acid had little influence on the rate or extent of growth of any of the strains except R. albus 7, for which the extent of growth was markedly increased. Determination of the quantitative fatty acid requirements for the three B. succinogenes strains indicated that 0.1 μmole of valeric per ml and 0.05 μmole of 2-methylbutyric per ml permitted maximal growth. However, with isobutyric acid as the branched-chain component, strains A3c and B21a required 0.1 μmole/ml in contrast to S-85 which exhibited optimal growth at the 0.05 μmole/ml level. By use of mixtures of isobutyric and 2-methylbutyric acids, good growth of C-94 was obtained at concentrations of 0.1 and 0.01 μmole/ml, respectively. About 0.3 μmole/ml of each acid was required for satisfactory growth of C1a.     

Detection of Coxiella burnetii in Ambient Air after a Large Q Fever Outbreak

One of the largest Q fever outbreaks ever occurred in the Netherlands from 2007–2010, with 25 fatalities among 4,026 notified cases. Airborne dispersion of Coxiella burnetii was suspected but not studied extensively at the time. We investigated temporal and spatial variation of Coxiella burnetii in ambient air at residential locations in the most affected area in the Netherlands (the South-East), in the year immediately following the outbreak. One-week average ambient particulate matter < 10 μm samples were collected at eight locations from March till September 2011. Presence of Coxiella burnetii DNA was determined by quantitative polymerase chain reaction. Associations with various spatial and temporal characteristics were analyzed by mixed logistic regression. Coxiella burnetii DNA was detected in 56 out of 202 samples (28%). Airborne Coxiella burnetii presence showed a clear seasonal pattern coinciding with goat kidding. The spatial variation was significantly associated with number of goats on the nearest goat farm weighted by the distance to the farm (OR per IQR: 1.89, CI: 1.31–2.76). We conclude that in the year after a large Q fever outbreak, temporal variation of airborne Coxiella burnetii is suggestive to be associated with goat kidding, and spatial variation with distance to and size of goat farms. Aerosol measurements show to have potential for source identification and attribution of an airborne pathogen, which may also be applicable in early stages of an outbreak.     

Aerosols of Mycoplasmas, L Forms, and Bacteria: Comparison of Particle Size, Viability, and Lethality of Ultraviolet Radiation

Aerosols of microorganisms were tested for particle size by use of an Andersen sampler. Mycoplasma aerosols had an average count median diameter (CMD) of 2.1 ± 0.5 μ. Staphylococcus aureus L forms gave an average CMD of 4.6 ± 1.7 μ; the diphtheroid L form, a CMD of 3.4 ± 0.3 μ. Escherichia coli had a CMD of 5.4 ± 2.5 μ; Neisseria sicca, 3.3 ± 0.5 μ; N. meningitidis, 3.4 ± 0.2 μ. S. aureus ATCC 6538, the parent strain of the L form, yielded a CMD of 3.9 ± 1.2 μ. Candida albicans gave an average CMD of 5.9 ± 1.4 μ. All organisms tested survived aerosolizing and could be recovered in viable form for at least 1 hr. Ultraviolet radiation at 2,537 A destroyed the bacteria and mycoplasmas instantaneously, and destroyed 87% of the L forms of S. aureus, 69% of the diphtheroid L form, and 98% of the C. albicans cells. After irradiation, viable particles of the L form and C. albicans aerosols were consistently larger, indicating that clumping led to survival. Submicron size particles were found in aerosols of all species tested except C. albicans.     

Physicochemical characterization of mitochondrial DNA from soybean

Mitochondrial DNA (mtDNA) of soybean (Glycine max L.) was isolated and its buoyant density was contrasted with that of nuclear (nDNA) and chloroplast (ctDNA) DNA. Each of the three DNAs banded at a single, characteristic buoyant density when centrifuged to equilibrium in a CsCl gradient. Buoyant densities were 1.694 g/cm³ for nDNA and 1.706 g/cm³ for mtDNA. These values correspond to G-C contents of 34.7 and 46.9%, respectively. Covalently closed, circular mtDNA molecules were isolated from soybean hypocotyls by ethidium bromide-cesium chloride density gradient centrifugation. Considerable variation in mtDNA circle size was observed by electron microscopy. There were seven apparent size classes with mean lengths of 5.9 μm (class 1), 10 μm (class 2), 12.9 μm (class 3), 16.6 μm (class 4), 20.4 μm (class 5), 24.5 μm (class 6), and 29.9 μm (class 7). In addition, minicircles were observed in all preparations. Partially denatured, circular mtDNA molecules with at least one representative from six of the seven observed size classes were mapped. In class 4, there appear to be at least three distinct denaturation patterns, indicating heterogeneity within this class. It is proposed that the mitochondrial genome of soybean is distributed among the different size circular molecules, several copies of the genome are contained within these classes and that the majority of the various size molecules may be a result of recombination events between circular molecules.     

Meloidogyne californiensis n. sp. (Nemata: Meloidogyninae), Parasitic On Bulrush,Scirpus robustus Pursh

Meloidogyne californiensis n. sp. is described and illustrated from bulrush Scirpus robustus in California. LM and SEM studies revealed that this species differs from other known species in the genus Meloidogyne especially by the prominent posterior cuticular protuberances in the female, the distinct shape of the perineal pattern which is marked by one prominent stria in the perineum, indistinct lateral lines, many broken discontinuous striae on both sides of the arch, and the excretory pore being located posterior to stylet base. Second-stage juveniles 448-628 μm long, stylet length 11-13 μm, styler delicate, with small knobs sloping posteriorly, cephalic region with 2 or 3 annuli, and inflated rectum. Males vary greatly in size (712-1,952 μm), stylet length 18-28 μm (mean 22 μm), cephalic region slightly set off the body with two or three annuli, spear heavy with massive rounded knobs, lateral field marked by four areolated incisures as seen by SEM.     

Respiratory Chain of Colorless Algae II. Cyanophyta

Whole cell difference spectra of the blue-green algae, Saprospira grandis, Leucothrix mucor, and Vitreoscilla sp. have one, or at the most 2, broad α-bands near 560 mμ. At −190° these bands split to give 4 peaks in the α-region for b and c-type cytochromes, but no α-band for a-type cytochromes is visible. The NADH oxidase activity of these organisms was shown to be associated with particulate fractions of cell homogenates. The response of this activity to inhibitors differed from the responses of the NADH oxidase activities of particulate preparations from the green algae and higher plants to the same inhibitors, but is more typical of certain bacteria. No cytochrome oxidase activity was present in these preparations. The respiration of Saprospira and Vitreoscilla can be light-reversibly inhibited by CO, and all 3 organisms have a CO-binding pigment whose CO complex absorbs near 570, 535, and 417 mμ. The action spectrum for the light reversal of CO-inhibited Vitreoscilla respiration shows maxima at 568, 534, and 416 mμ. The results suggest that the terminal oxidase in these blue-greens is an o-type cytochrome.     

The Effect of C. burnetii Infection on the Cytokine Response of PBMCs from Pregnant Goats

In humans, infection with Coxiella burnetii, the causative agent of Q fever, leads to acute or chronic infection, both associated with specific clinical symptoms. In contrast, no symptoms are observed in goats during C. burnetii infection, although infection of the placenta eventually leads to premature delivery, stillbirth and abortion. It is unknown whether these differences in clinical outcome are due to the early immune responses of the goats. Therefore, peripheral blood mononuclear cells (PBMCs) were isolated from pregnant goats. In total, 17 goats were included in the study. Six goats remained naive, while eleven goats were infected with C. burnetii. Toll-like receptor (TLR) and cytokine mRNA expression were measured after in vitro stimulation with heat-killed C. burnetii at different time points (prior infection, day 7, 35 and 56 after infection). In naive goats an increased expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-10 and interferon (IFN)-γ mRNA upon C. burnetii stimulation was detected. In addition, TLR2 expression was strongly up-regulated. In goats infected with C. burnetii, PBMCs re-stimulated in vitro with C. burnetii, expressed significantly more TNF-α mRNA and IFN-γ mRNA compared to naive goats. In contrast, IL-10 mRNA production capacity was down-regulated during C. burnetii infection. Interestingly, at day 7 after inoculation a decreased IFN-γ protein level was observed in stimulated leukocytes in whole blood from infected goats, whereas at other time-points increased production of IFN-γ protein was seen. Our study shows that goats initiate a robust pro-inflammatory immune response against C. burnetii in vitro. Furthermore, PBMCs from C. burnetii infected goats show augmented pro-inflammatory cytokine responses compared to PBMCs from non-infected goats. However, despite this pro-inflammatory response, goats are not capable of clearing the C. burnetii infection.     

Production of Sterigmatocystin by Some Species of the Genus Aspergillus and Its Toxicity to Chicken Embryos

Sterigmatocystin was produced by 59% of Aspergillus flavus cultures and by 16% of A. parasiticus cultures. All sterigmatocystin-producing cultures of the A. flavus group also simultaneously produced aflatoxin or O-methylsterigmatocystin. Sterigmatocystin was produced by A. chevalieri, A. ruber, and A. amstelodami, species not previously reported to produce the compound. In 5-day-old chicken embryos, the no-effect level of toxicity of sterigmatocystin was between 1 and 2 μg/egg; the mean lethal dose was 5 to 7 μg; and 90 to 100% of the embryos were killed with 10 μg. Teratogenic effects and weight reduction were generally associated with nonlethal doses.     

Antibodies Are Required for Complete Vaccine-Induced Protection against Herpes Simplex Virus 2

Herpes simplex virus 2 (HSV-2) 0ΔNLS is a live HSV-2 ICP0 ^- mutant vaccine strain that is profoundly attenuated in vivo due to its interferon-hypersensitivity. Recipients of the HSV-2 0ΔNLS vaccine are resistant to high-dose HSV-2 challenge as evidenced by profound reductions in challenge virus spread, shedding, disease and mortality. In the current study, we investigated the requirements for HSV-2 0ΔNLS vaccine-induced protection. Studies using (UV)-inactivated HSV-2 0ΔNLS revealed that self-limited replication of the attenuated virus was required for effective protection from vaginal or ocular HSV-2 challenge. Diminished antibody responses in recipients of the UV-killed HSV-2 vaccine suggested that antibodies might be playing a critical role in early protection. This hypothesis was investigated in B-cell-deficient μMT mice. Vaccination with live HSV-2 0ΔNLS induced equivalent CD8⁺ T cell responses in wild-type and μMT mice. Vaccinated μMT mice shed ~40-fold more infectious HSV-2 at 24 hours post-challenge relative to vaccinated wild-type (B-cell⁺) mice, and most vaccinated μMT mice eventually succumbed to a slowly progressing HSV-2 challenge. Importantly, passive transfer of HSV-2 antiserum restored full protection to HSV-2 0ΔNLS-vaccinated μMT mice. The results demonstrate that B cells are required for complete vaccine-induced protection against HSV-2, and indicate that virus-specific antibodies are the dominant mediators of early vaccine-induced protection against HSV-2.     

Resistance of Pseudomonas aeruginosa to gentamicin

The resistance to gentamicin 4 μg./ml. of 250 Pseudomonas aeruginosa isolates was measured by a proportion method. Twenty-eight (11.2%) of the cultures fell into the most resistant group, in whose populations between 10 and 100% of the organisms were resistant. A relatively high percentage of urinary isolates and a comparatively low percentage of isolates from respiratory sources occurred in this group. Three of the 28 were resistant to carbenicillin 150 μg./ml. and 6 of 18 tested were as resistant to gentamicin 8 μg./ml. as they were to 4 μg./ml. The distribution of Ps. aeruginosa isolates between the different grades of resistance did not change significantly during the 10 months in which the survey was performed.     

Mycobacterium gilvum Illustrates Size-Correlated Relationships between Mycobacteria and Acanthamoeba polyphaga

Mycobacteria are isolated from soil and water environments, where free-living amoebae live. Free-living amoebae are bactericidal, yet some rapidly growing mycobacteria are amoeba-resistant organisms that survive in the amoebal trophozoites and cysts. Such a capacity has not been studied for the environmental rapidly growing organism Mycobacterium gilvum. We investigated the ability of M. gilvum to survive in the trophozoites of Acanthamoeba polyphaga strain Linc-AP1 by using optical and electron microscopy and culture-based microbial enumerations in the presence of negative controls. We observed that 29% of A. polyphaga cells were infected by M. gilvum mycobacteria by 6 h postinfection. Surviving M. gilvum mycobacteria did not multiply and did not kill the amoebal trophozoites during a 5-day coculture. Extensive electron microscopy observations indicated that M. gilvum measured 1.4 ± 0.5 μm and failed to find M. gilvum organisms in the amoebal cysts. Further experimental study of two other rapidly growing mycobacteria, Mycobacterium rhodesiae and Mycobacterium thermoresistibile, indicated that both measured <2 μm and exhibited the same amoeba-mycobacterium relationships as M. gilvum. In general, we observed that mycobacteria measuring <2 μm do not significantly grow within and do not kill amoebal trophozoites, in contrast to mycobacteria measuring >2 μm (P < 0.05). The mechanisms underlying such an observation remain to be determined.     

Enhanced Growth and Activity of a Biocontrol Bacterium Genetically Engineered To Utilize Salicylate

Plasmid NAH7 was transferred from Pseudomonas putida PpG7 to P. putida R20 [R20(NAH7)], an antagonist of Pythium ultimum. The plasmid did not affect growth or survival of R20(NAH7) and was stably maintained under nonselective conditions in broth and soil and on sugar beet seeds. Plasmid NAH7 conferred to R20(NAH7) the ability to utilize salicylate in culture, agricultural field soil, and on sugar beet seeds. The metabolic activity of R20(NAH7), but not the wild-type R20, was greatly increased in soil by amendment with salicylate (250 μg/g) as measured by induced respiration. Population densities of R20(NAH7) were also enhanced in salicylate-amended soil, increasing from approximately 1 × 10⁵ CFU/g to approximately 3 × 10⁸ CFU/g after 35 h of incubation. In contrast, population densities of R20(NAH7) in nonamended soil were approximately 3 × 10⁶ CFU/g of soil after 35 h of incubation. The concentration of salicylate in soil affected the rate and extent of population increase by R20(NAH7). At 50 to 250 μg of salicylate per g of soil, population densities of R20(NAH7) increased to approximately 10⁸ CFU/g of soil by 48 h of incubation, with the fastest increase at 100 μg/g. A lag phase of approximately 24 h occurred before the population density increased in the presence of salicylate at 500 μg/g; at 1,000 μg/g, population densities of R20(NAH7) declined over the time period of the experiment. Population densities of R20(NAH7) on sugar beet seeds in soils amended with 100 μg of salicylate per g were not increased while ample carbon was present in the spermosphere. However, after carbon from the seed had been utilized, population densities of R20(NAH7) decreased significantly less (P = 0.005) on sugar beet seeds in soil amended with salicylate than in nonamended soil.