Microalgae response to petroleum spill: an experimental model analysing physiological and genetic response of Dunaliella tertiolecta (Chlorophyceae) to oil samples from the tanker Prestige.

In November 2002, the oil tanker Prestige sank off the northwestern coast of Spain, spilling more than 50,000 tons of petroleum with disastrous ecological and economical consequences. In order to analyse the harmful consequences of the oil spill on marine microalgae, short- and long-term effects of oil samples from the Prestige spill were studied using laboratory cultures of Dunaliella tertiolecta (strain Dt1Lwt). Significant inhibition of photosynthesis (assessed by F(v)/F(m), ETR(max) and alpha estimations) was observed after only 1h of oil exposure with clear concentration dependency. Three days later, photosynthetic activity remained inhibited although cell survival was only slightly effected. In cultures exposed to the lowest oil concentration, mitotic rates and percentage of motile cells were 17-33% and 12-42% of the controls, respectively. After 1 month, neither dividing nor motile cells were observed at the highest oil concentrations. However, after further incubation, occasionally the growth of rare cells resistant to oil was found. A fluctuation analysis was carried out to distinguish between resistant cells arising from rare spontaneous mutations and resistant cells arising from physiological or other mechanisms of adaptation. The existence of rapid evolution as result of preselective mutations from petroleum sensitivity to petroleum resistance was observed. Resistant cells arose by rare spontaneous mutations prior to the addition of oil, with a mutation rate of 2.76x10(-5) oil-resistant mutants per cell division. Apparently, rare spontaneous preselective mutations are able to assure the survival of microalgae in oil-polluted environments.

Effects of re-infection with Neospora caninum in bulls on parasite detection in semen and blood and immunological responses.

Three bulls with experimentally induced primary infection with Neospora caninum were re-infected intravenously with 10(8) live N. caninum tachyzoites of the NC-1 isolate at 300 days post-infection to investigate the presence of N. caninum in semen and blood, and the associated immune responses. In parallel, three bulls with experimentally induced primary infection with N. caninum and three non-infected bulls were also monitored. Re-infected and infected bulls showed an intermittent presence of N. caninum DNA in semen with a parasite load ranging from 0.1 to 15.6 (mean 4.4) and 0.1 to 11.1 (mean 4.1) parasites/ml, respectively. Re-infected bulls showed significant and persistent serum-specific IgM and IgG antibody responses. Specific IgG levels were detected in seminal plasma of all infected bulls, but the magnitude of the response was significantly higher in re-infected rather than in chronically infected animals. The mean specific IFN-gamma levels in re-infected bulls were significantly increased as early as 3 and 7 days after experimental infection when compared to bulls in other groups. This study showed that the intermittent presence and parasite load of N. caninum in the semen of re-infected bulls is very similar to that reported in chronically infected animals. The protozoa could not be isolated from BALB/c nu/nu mice inoculated with PCR-positive semen samples and inseminated heifers with pooled semen samples did not show seroconversion. Plasma IFN-gamma level seems to be a good indicator of a recent N. caninum infection.

Experimental neosporosis in bulls: parasite detection in semen and blood and specific antibody and interferon-gamma responses.

AIM: To investigate the presence of Neospora caninum in semen and blood, and the development of specific antibody and interferon-gamma (IFN-gamma) responses in experimentally infected bulls. METHODS: Eight bulls were intravenously infected with 10(8) live N. caninum tachyzoites of NC-1 isolate. The presence of N. caninum in semen and blood was assessed using a nested-PCR procedure. PCR-positive semen samples were bioassayed using a BALB/c nu/nu mouse model. Specific anti-N. caninum antibody and IFN-gamma responses were also examined. In parallel, eight seronegative bulls were studied as non-infected controls. All bulls were monitored for 26 weeks. RESULTS: All eight experimentally infected bulls showed N. caninum DNA in their semen and/or blood samples at some time during the course of the study. Parasite load in semen ranged from 0.1 to 14.5 parasites/ml (mean 6.0). N. caninum could not be detected in BALB/c nu/nu mice inoculated with PCR-positive semen samples. A significant increase in mean serum specific IgM antibody response to N. caninum was detected between 10 and 28 days post-infection (p.i.). Serum specific IgG, IgG1, and IgG2 antibody levels in experimentally infected bulls were significantly different after 21, 10, and 14 days p.i. as compared to controls, respectively. Specific anti-N. caninum IgG were detected in seminal plasma from infected bulls and values obtained were different from controls after 25 days p.i. Mean specific IFN-gamma responses in experimentally infected bulls were significantly higher than controls 3 days p.i. CONCLUSIONS: This is the first study to report the presence of N. caninum DNA in the semen and blood of experimentally infected bulls. Our observations indicate an intermittent presence of N. caninum in low numbers in semen and associated with chronic stage of the infection. This study is also the first to report the detection of anti-N. caninum IgG in seminal plasma of experimentally infected bulls.

Anthelmintic and nutritional effects of heather supplementation on Cashmere goats grazing perennial ryegrass-white clover pastures.

To investigate anthelmintic and nutritional effects of heather supplementation in goats grazing perennial ryegrass-white clover pastures, 40 dry Cashmere goats were randomly assigned to 4 treatments in a 2 x 2 factorial arrangement: 2 grazing management treatments (supplementation with heather vs. nonsupplementation) and 2 anthelmintic treatments (treatment vs. nontreatment). Goats grazed continuously from May to September 2004. At the end of the grazing period, the number of dead goats due to gastrointestinal parasitism was 1 in the group supplemented with heather and dosed with anthelmintic, 4 in the group that received neither supplementation nor anthelmintic, and 0 in the other 2 groups. For goats that did not receive anthelmintic treatment, the percentage of heather in the diet was negatively correlated with fecal egg count in August (r = -0.59, P < 0.05) and September (r = -0.49, P < 0.1) and positively correlated (r = 0.54, P < 0.05) with BW changes during the grazing season. Therefore, the correlation coefficient between BW change and fecal egg count was negative (r = -0.62, P < 0.05). Rumen ammonia concentrations were always lower in supplemented goats (P < 0.05). However, VFA concentrations were greater in goats consuming heather (58.9 vs. 50.9 mmol/L), which suggests that ruminal fermentation was not adversely affected by consumption of tannins. Heather availability in the vegetation might represent a valuable opportunity and sustainable method to control gastrointestinal nematode infections in a goat production system based on grazing perennial ryegrass-white clover pastures.

Intrauterine Neospora caninum inoculation of heifers.

Here, we studied the potential of Neospora caninum tachyzoites to infect heifers when administered in utero by artificial insemination via contaminated semen. Eighteen primiparous cyclic heifers were hormonally synchronized and artificially inseminated. Nine of them, which were inseminated with semen containing 10(7) live N. caninum NC-1 isolate-tachyzoites, reacted with seroconversion and a specific IFN-gamma response. Moreover, N. caninum DNA was demonstrated by a nested-PCR in the blood of all nine heifers and in brain, lungs, liver and uterine horn of several of them. In contrast, nine heifers inseminated with tachyzoite-free semen developed no antibody or IFN-gamma responses, and no parasite DNA was detected in blood or organs. At necropsy, viable embryos were detected in one and six of the infected and non-infected heifers, respectively. No specific Neospora DNA was detected in any of the embryos. This study provides evidence that intrauterine inoculation via contaminated semen cause N. caninum infection in cattle.