Selection of Specific Endophytic Bacterial Genotypes by Plants in Response to Soil Contamination

Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants. At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (ntdAa) and nitrotoluene monooxygenase (ntnM), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of ndoB-positive endophytes in Scirpus pungens, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants. Similarly, the numbers of alkB- or ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not with the numbers of alkB- or ndoB-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.     

Genetic modifications of pigs for medicine and agriculture

Genetically modified swine hold great promise in the fields of agriculture and medicine. Currently, these swine are being used to optimize production of quality meat, to improve our understanding of the biology of disease resistance, and to reduced waste. In the field of biomedicine, swine are anatomically and physiologically analogous to humans. Alterations of key swine genes in disease pathways provide model animals to improve our understanding of the causes and potential treatments of many human genetic disorders. The completed sequencing of the swine genome will significantly enhance the specificity of genetic modifications, and allow for more accurate representations of human disease based on syntenic genes between the two species. Improvements in both methods of gene alteration and efficiency of model animal production are key to enabling routine use of these swine models in medicine and agriculture.     

Close synchrony of ovulation in superstimulated heifers that have a downregulated anterior pituitary gland and are induced to ovulate with exogenous LH

The synchrony of ovulation was examined in superstimulated heifers that had a downregulated pituitary gland and which were induced to ovulate by injection of exogenous LH. The pituitary was downregulated and desensitized to GnRH by treatment with the GnRH agonist deslorelin. Nulliparous heifers (3.5 yr old) at random stages of the estrous cycle were assigned to 1 of 3 groups, and on Day -7 received the following treatments: Group 1 (control, n = 8), 1 norgestomet ear implant; Group 2 (GnRH agonist, n = 8); Group 3 (GnRH agonist-LH protocol, n = 8), 2 deslorelin ear implants. Ovarian follicle growth in all heifers was superstimulated with twice-daily intramuscular injections of FSH (Folltropin-V): Day O, 40 mg (80 mg total dose); Day 1, 30 mg; Day 2; 20 mg; Day 3, 10 mg. On Day 2, all heifers were given a luteolytic dose of PGF (7 A.M.), Norgestomet implants were removed from heifers in Group 1 (6 P.M.). Heifers in Group 3 were given an injection of 25 mg, i.m. porcine LH (Lutropin) on Day 4 (4 P.M.). Ovarian follicle status was monitored at 8-h intervals from Day 3 (8 A.M.) to Day 6 (4 P.M.) using an Aloka Echo Camera and 7.5 MHz transducer. Heifers in Groups 2 and 3 exhibited estrus earlier (P < 0.05) than heifers in Group 1. Heifers in Group 2 did not have a preovulatory LH surge and they did not ovulate. Individual control heifers in Group 1 ovulated between 12 A.M. on Day 5 and 8 A.M. on Day 6. Heifers with deslorelin implants and injected with LH in Group 3 ovulated between 4 P.M. on Day 5 and 8 A.M. on Day 6. It was confirmed that superstimulated heifers with GnRH agonist implants can be induced to ovulate with LH. It was also demonstrated that ovulation is closely synchronized after injection of LH. Thus, a single, fixed-time insemination schedule could be used in a GnRH agonist-LH superovulation protocol, with significant practical and economic advantages for superovulation and embryo transfer programs.     

Sex-specific changes in platelet aggregation and secretion with sexual maturity in pigs.

Cardiovascular disease may begin early in adolescence. Platelets release factors contributing to vascular disease. Experiments were designed to test the hypothesis that hormonal transitions associated with sexual maturity differentially affect platelet aggregation and secretion in males and females. Platelets were collected from juvenile (2-3 mo) and sexually mature (adult; 5-6 mo) male and female pigs (n=8/group). Maturation was evidenced by increased weight of reproductive tissue and changes in circulating levels of gonadal hormones. Aggregation to ADP (10 microM) and collagen (6 microg/ml) and ATP secretion to 50 nM thrombin were determined by turbidimetric analysis and bioluminescence, respectively. Total platelet counts, platelet turnover, and mean platelet volume did not change with maturity. Platelet aggregation and ATP secretion decreased in females but increased in males with maturity, whereas total ATP content remained unchanged in platelets from females but increased in platelets from males. Platelet fibrinogen receptor, P-selectin expression, and receptors for sex steroids did not change with sexual maturation. Plasma C-reactive protein and brain-type natriuretic peptide also did not change. Results indicate that changes in platelet aggregation and secretion change with sexual maturity differently in females and males. These observations provide evidence on which clinical studies could be designed to examine platelet characteristics in human children and young adults.     

Biodegradation of petroleum hydrocarbons by psychrotrophic Pseudomonas strains possessing both alkane (alk) and naphthalene (nah) catabolic pathways.

Three hydrocarbon-degrading psychrotrophic bacteria were isolated from petroleum-contaminated Arctic soils and characterized. Two of the strains, identified as Pseudomonas spp., degraded C5 to C12 n-alkanes, toluene, and naphthalene at both 5 and 25 degrees C and possessed both the alk catabolic pathway for alkane biodegradation and the nah catabolic pathway for polynuclear aromatic hydrocarbon biodegradation. One of these strains contained both a plasmid slightly smaller than the P. oleovorans OCT plasmid, which hybridized to an alkB gene probe, and a NAH plasmid similar to NAH7, demonstrating that both catabolic pathways, located on separate plasmids, can naturally coexist in the same bacterium.     

Bioremediation Assessment of Hydrocarbon-Contaminated Soils from the High Arctic

The bioremediation potential of hydrocarbon-contaminated soils from the most northerly inhabited station in the world, Canadian Forces Station - Alert, was assessed. Microbial enumeration, by both viable plate counts and direct counts, combined with molecular analysis (polymerase chain reaction and colony hybridization) for hydrocarbon catabolic genes (alkB, ndoB, xylE), demonstrated the presence of significant numbers of cold-adapted hydrocarbon-degrading microorganisms. The degradative activity of these populations was assessed by mineralization of ¹⁴Clabeled hexadecane (C16) at 5°C in untreated and treated soils. Although very low rates of C16 mineralization were observed in the untreated soils, nutrient supplementation with a fertilizer markedly increased C16 mineralization. Highly active cold-adapted hydrocarbon-degrading consortia were prepared from soil slurries, and their degradative potentials were monitored by biomass measurements and mineralization activity. Bio augmentation of the contaminated soils with consortia containing the greatest percentages of degradative bacteria resulted in the shortest C16 mineralization acclimation period. However, treatment with the consortia plus fertilizer did not appreciably increase C16 mineralization or reduce total petroleum hydrocarbon concentrations to a greater extent than did the fertilizer treatment alone. These results indicate that the soils possessed sufficient numbers of cold-adapted degradative bacteria, and that fertilizer application alone was sufficient to obtain elevated levels of degradative activity at low ambient summer temperatures.     

Transformation of a psychrotrophic bacterium with a plasmid from a mesophile

The psychrotrophic bacteriumBacillus psychrophilus was transformed with the broadhost-range plasmid pC194. The ability of the transformant to express chloramphenicol (CAM) resistance and the possible effects of such expression on the physiology of the psychrotroph were examined. The transformant exhibited growth rates, filament formation at elevated temperatures, synthesis of cold shock proteins and cold acclimation proteins, similar to the parentalB. psychrophilus.     

Contraceptive efficacy of an intra-uterine device in Brahman cattle

The contraceptive efficacy of an intra-uterine device was evaluated using 218 heifers and 212 cows on three north Australian cattle stations. The heifers were aged approximately 2 years and weighed 250-378 kg; the cows were aged 3-16 years and weighed 256-540 kg. All cattle were non-pregnant, non-lactating Brahmans. At the end of the monsoon (wet) season (April-June 1997), the cattle were allocated by stratified randomisation to the three treatments which were untreated controls (n=59), surgical ovariectomy (n=105), or implantation with a bovine intra-uterine device (BIUD; n=266). All cattle grazed and were managed as one group within each station. They were exposed to bulls (4 per 100 females) from soon after treatment until slaughter approximately 12 months later.The BIUD could not be implanted in 25% of heifers and 8% of cows due to narrow or twisted cervices. Correct placement of the BIUDs appeared to be achieved in 57% of heifers and 72% of cows. At slaughter, the devices were incorrectly positioned in 73% of heifers and 49% of cows into which BIUDs had been inserted and that remained non-pregnant. Uterine perforations by the BIUD were observed in 35 and 45% of these heifers and cows, respectively; most perforations appeared to occur during implantation. Low-grade endometritis was observed at slaughter in most BIUD-implanted animals; 2% had pyometra.BIUD animals did not have significantly different growth to that of control or ovariectomised animals, other than when ovariectomy suppressed growth following surgery. Most animals implanted with BIUDs appeared to have normal ovarian function and animals were observed mating. All ovariectomised animals remained non-pregnant. Over 80% of controls were pregnant within 8 months of exposure to bulls, except heifers at one station where pregnancy rate was restricted to 25% as a result of severe nutritional conditions. Pregnancy was diagnosed in 21% of heifers and 33% of cows with implanted BIUDs. The device remained correctly positioned and with no pregnancy diagnosed in the year following implantation in only 2% of heifers and 14% of cows originally allocated.Because of the difficulties of implanting BIUDs, the high frequency of associated uterine injury, the high pregnancy rate in implanted animals, and that growth was unaffected by the presence of a BIUD, it was concluded that the device had poor contraception efficacy and no growth-promotant effect in Brahman cattle.