In vitro modulation of reactive oxygen and nitrogen intermediate (ROI/RNI) production in Crassostrea gigas hemocytes

Bivalve hemocyte competence has been measured by quantifying functional characteristics, including reactive oxygen intermediate (ROI) production after activation with zymosan or phorbol myristate acetate (PMA). However, untreated oyster hemocytes also produce ROI and RNI (reactive nitrogen intermediates) after bleeding even if not stimulated by zymosan or PMA. Extensive investigation of this parameter by flow cytometry showed that, in vitro, ROI/RNI production by untreated hemocytes maintained in seawater appeared to be independent of both bacterial burden in the serum and non-self particle phagocytosis. ROI/RNI production in granulocytes was higher than in hyalinocytes and could be intensified when activated by zymosan but not by PMA. Both cell types used NADPH-oxidase- and NO-synthase-like pathways to produce these molecules; the NO-synthase pathway seemed relatively more dominant in hyalinocytes and NADPH-oxidase appeared more effective in granulocytes. These results provide new insights for interpreting the modulation of ROI/RNI production by untreated hemocytes shown by other studies, relative to environmental conditions or physiological status of the oysters.     

Soluble sugars and carboxylic acids in ripe apricot fruit as parameters for distinguishing different cultivars

Major sugar and carboxylic acid components in apricot flesh fruits were detected and quantified. Fifty-one genotypes including clones growing in France, Spain, Italy, Greece and USA, belonging to the INRA germ plasm collection has been evaluated. Principal component analysis (PCA) has been performed to study correlation among fruit quality measurements and to interpret relationships between genotypes as a tool for germ plasm characterization. A marked variation in malic and citric acid content has been observed and PCA revealed clusters of apricot genotypes for the malic/citric ratio content. A good correlation between sugar component and refractometer index (r = 0.83) has been detected. The wide range of diversity in malic and citric acid content in apricot germ plasm makes it possible to breed and select genotypes with improved flavour on the basis of superior phenotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Exposure Patterns and Long Term Sequelae on Adults and Children in Two Canadian Indigenous Communities Exposed to Methylmercury

Health Canada has been collecting data on Inuit and First Nations' methylmercury (MeHg) levels for 25 years. A national overview has been completed and more focussed analyses have now been initiated. This paper deals with two interdependent analytical components: 1) a longitudinal overview of the two most extensively sampled communities in Canada: Grassy Narrows and Whitedog, the residents of which were exposed to ‘point source’ mercury pollution in the 1970's; 2) fetal and post-natal exposure to mercury in these two communities including an outline of the First Nation MeHg child development pilot project in the two communities. A retrospective analysis of Grassy Narrows and Whitedog shows a decreasing MeHg trend in both communities. In Grassy Narrows the average individual annual peak methylmercury level in blood has decreased significantly, from 23.80 ppb (range 1.50–322.90) in 1976 to 7.5 ppb (1.7–46.7) in 1995 (r=-0.65, p<0.001). In Whitedog the average peak has also decreased significantly, from 12.87 ppb (1.50–172.00) in 1976 to 6.1 ppb (1.7–33.3) in 1995 (r=-0.59, p<0.005). However, behind these positive trends is the reality of two communities still suffering the effects of disrupted lifestyles and socio-cultural damage. A number of cord blood samples, maternal blood samples, and samples from women of child-bearing age from these communities have been in the “risk” group (according to the 1990 WHO guidelines). We are therefore now assessing the long term effects of fetal exposure in the communities. Standard clinical examinations in the past failed to prove abnormalities attributable to methylmercury but did not include subtle neuropsychological development tests. Many of the fetally exposed children are now in secondary school. Therefore, we have initiated a pilot project to assess long term effects of methylmercury exposure on the neuropsychological development of these children whose fetal exposure we know. The pilot child development project which was initiated in 1995 in Grassy Narrows and Whitedog, with community support, includes four main components: i) a school records review, looking at attendance, marks, and atypical behaviour; ii) teachers' questionnaires, targeting child behaviour; iii) an in-depth neuropsychological test battery focussing on subtle factors, such as memory, attention, executive functions, perceptual functions and sensory / motor development; and iv) hair sampling, providing current MeHg levels to correlate with results from the previous sampling and findings from the first three components. An overview of progress is given.     

Pomological and Nutraceutical Properties in Apricot Fruit: Cultivation Systems and Cold Storage Fruit Management

We have investigated the effect of cultivation systems and fruit post-harvest management on the antioxidant properties of apricot fruits. Trees of five cultivars ‘Tyrinthos’, ‘Cafona’, ‘Bella d’Italia’, ‘Vitillo’ and ‘Pellecchiella’ were cultivated under integrated and organic systems. Fruits were collected at full maturity stage and analyzed either immediately or after storage at 4±0.5 °C and 85% of relative humidity for seven and 14 days. The main pomological traits (weight, colour, flesh firmness, total soluble sugars, titratable acidity) and antioxidant properties were analyzed. The total antioxidant capacity (TAC by TEAC method), total phenols content (TP by Folin-Ciocalteu method) and carotenoid content by HPLC-DAD were monitored. Cultivar characterization by principal component analysis (PCA) indicated a large variability on pomological and antioxidant properties of apricot fruits. ‘Bella d’Italia’ showed better TAC and TP values compared to the other cultivars. ANOVA interactions between cultivar and cultivation system (organic/integrated) were found for the antioxidant properties. These interactions may help to select a set of genotypes with better performances under organic system, which in our study might be indicated in ‘Cafona’ and ‘Bella d’Italia’.     

Hemocyte characteristics in families of oysters, Crassostrea gigas, selected for differential survival during summer and reared in three sites

High variability among individuals is often encountered when hemocyte characteristics are measured in bivalves. Such variability is suspected to result partly from genetic factors. In this study, hemocyte characteristics of six families of Crassostrea gigas were compared by flow cytometry at one sampling date in October 2001. These families were obtained from a nested, half-sibling cross design, and reared from July to October 2001 at three sites distributed along the French Atlantic coast from north to south: Baie des Veys (Normandy), Rivière d'Auray (Brittany) and Ronce (Marennes-oléron Basin, Poitou Charentes).

Among the 15 measured hemocyte characteristics, production of reactive oxygen species (ROS) of untreated hemocytes (maintained in filtered sterile seawater) and treated hemocytes (zymosan at 20 particles per hemocyte, and with Vibrio sp. S322 at 50 bacteria per hemocyte) was the most notable differences between families. This supports the existence of a genetic basis, at least partly, for the hemocyte characteristics of oysters, and especially for ROS production.

Among the six families analyzed, three have shown high survival during summer (named as “resistant”, mean mortality 5.2%) and three experienced high mortality during summer (named as “susceptible”, 30.6% mean mortality). Families showing high or low survival to summer mortality had similar hemocyte characteristics, regardless of the environmental conditions or reproductive state. Resistant families were observed to have higher total hemocyte counts and lower production of ROS than susceptible families. Moreover, ROS production of hemocytes from susceptible families was diminished significantly more by pathogenic Vibrio than that of resistant families. However, this study demonstrates also that rearing site strongly affected the hemocyte characteristics of all families of oysters, most notably hemocyte concentration and morphology (size and granularity), production of reactive oxygen species (ROS), and susceptibility to the cytotoxic activity of the pathogenic Vibrio sp. S322 (50 bacteria/hemocyte). Food availability and reproductive state are the most probable explanations for the site differences observed. Finally, it appeared difficult to link oyster survival during summer mortality to hemocyte profiles evaluated at one sampling date; other relevant indicators would probably help explaining oyster survival during summer mortality events.     

Modelling nitrogen stress with probe genotypes to assess genetic parameters and genetic determinism of winter wheat tolerance to nitrogen constraint

Environmental and economical constraints in Europe will favour low nitrogen (N) input systems and wheat varieties adapted to moderate N deficiency. In this context, we studied the dynamics of genetic parameters according to N stress intensity and characterized the genetic determinants for plant tolerance to N deficiency. Thus, we combined N stress modelling with a genetic approach. Two hundred and twenty-two doubled haploid lines were experimented in the field for a range of nitrogen conditions. Those conditions were characterized by the Nitrogen Nutrition Index (NNI) of Récital. Grain Yield (GY) and Kernel Number (KN) were assessed. For GY and KN, and for each line, factorial regressions using NNI of Récital as environmental index were performed. In addition, we assessed the sensitivity to N stress (slopes of the regression) and the performances under low N conditions (predicted values for a NNI of 0.5). QTL detection was performed on these parameters as well as on KN and GY measured in each environment. G × N variance increased with N stress intensity whereas heritability and genetic variance decreased. 11 QTL regions were detected: 3 were N supply-specific QTL (on linkage groups 2A2, 3A and 4B) while 4 contained QTL detected under N⁺ and under N⁻ (2D1, 4B and 5A1). Out of these four, 2 coincided with QTL for factorial regression parameters (2D1 and 4B). Finally, 4 QTL were specific for factorial regression parameters (3B, 5A2 and 7B2). The role of genes commonly used in breeding programs (rht-B1 on 4B, and Ppd1 on 2D1) in plant adaptation to nitrogen constraint was highlighted. Future studies should focus on grain protein yield, another target for low-N breeding scheme.     

Mic1-3 Knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep

This study assessed the effectiveness of a mutant strain of Toxoplasma gondii (RH strain) lacking the mic1 and mic3 genes (Mic1-3KO) against Toxoplasma abortion in sheep. Ewes were inoculated subcutaneously with 10⁵ Mic1-3KO tachyzoïtes in three independent experiments. Following vaccination, Mic1-3KO induced a mild febrile response and serum IgG antibodies, which persisted throughout the experiments. Tissue cysts formed in the sheep, but were not, under our experimental conditions, infectious when given orally. Ewes were mated two months after vaccination and were orally challenged with the PRU strain of T. gondii at mid-gestation (400 oocysts in Experiments 1 and 2; 100 oocysts in Experiment 3). Challenge of vaccinated pregnant ewes resulted in a slight febrile response, whereas unvaccinated ewes developed a more severe, characteristic febrile response of longer duration. After challenge, all unvaccinated ewes aborted whereas 62%, 91% and 64% (Experiments 1, 2 and 3 respectively) of the lambs from vaccinated ewes were viable, with no clinical signs of infection. Mic1-3KO was as effective as S48, the strain used as a live vaccine for sheep (Toxovax^®). A dose of 10⁵ Mic1-3KO tachyzoites was sufficient to induce protection (versus a dose of 2 × 10⁶). Both subcutaneous and intraperitoneal injections were effective. Moreover, preliminary results showed the potential of Mic1-3KO to reduce the development of tissue cysts in lambs born to vaccinated ewes. This study demonstrates that Mic1-3KO is a potent vaccine candidate.     

Urinary acidification in the prevention and treatment of feline struvite urolithiasis

Two groups of 12 cats were fed ad libitum an experimental dry expanded ration high in magnesium (0.37%, dry matter basis) and similar to most commercial dry cat foods in all other nutrients. The diet of one of the groups contained 1.5% ammonium chloride, which was added to the diet before processing. Urethral obstruction developed twice in 7 of the 12 cats fed the ration without ammonium chloride. Of the 5 remaining cats in that group, 2 had calculi in the urinary bladder and urethra at necropsy. In contrast, only 2 of the 12 cats fed the ration containing ammonium chloride had urethral blockage once; neither cat had another blockage. Both cats had been catheterized twice as paired controls before urethral obstruction. One of these 2 cats died from a urinary tract infection apparently introduced through the indwelling catheter, and at necropsy there were 2 small calculi in the urethra. At the end of the study, only 1 other cat fed the ammonium chloride diet had a calculus in the urinary bladder at necropsy; this cat had not had a urethral obstruction throughout the study. After each cat had 2 urethral blockages, its diet was changed. Four cats were fed the ration containing ammonium chloride and 3, a dry, commercial ration. Of the 4 cats fed the diet containing ammonium chloride, none developed another urethral obstruction within 6 months, and at necropsy, no calculi were in the urinary bladder or urethra. Two of the 3 cats fed the commercial ration experienced urethral blockage within 3 months. These 2 cats were then fed the diet containing ammonium chloride.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of metal-phosphates formation in the rhizosphere soils of pea and tomato: environmental and sanitary consequences

Purpose

At the global scale, soil contamination with persistent metals such as lead (Pb), zinc (Zn), and copper (Cu) induces a serious threat of entering the human food chain. In the recent past, different natural and synthetic compounds have been used to immobilize metals in soil environments. However, the mechanisms involved in amendment-induced immobilization of metals in soil remained unclear. The objective of the present work was therefore to determine the mechanisms involved in metal-phosphates formation in the rhizospheric soils of pea and tomato currently cultivated in kitchen gardens.

Materials and methods

Pea and tomato were cultivated on a soil polluted by past industrial activities with Pb and Zn under two kinds of phosphate (P) amendments: (1) solid hydroxyapatite and (2) KH₂PO₄. The nature and quantities of metal-P formed in the rhizospheric soils were studied by using the selective chemical extractions and employing the combination of X-ray fluorescence micro-spectroscopy, scanning electron microscopy, and electron microprobe methods. Moreover, the influence of soil pH and organic acids excreted by plant roots on metal-P complexes formation was studied.

Results and discussion

Our results demonstrated that P amendments have no effect on metal-P complex formation in the absence of plants. But, in the presence of plants, P amendments cause Pb and Zn immobilization by forming metal-P complexes. Higher amounts of metal-P were formed in the pea rhizosphere compared to the tomato rhizosphere and in the case of soluble P compared to the solid amendment. The increase in soil-metal contact time enhanced metal-P formation.

Conclusions

The different forms of metal-P formed for the different plants under two kinds of P amendments indicate that several mechanisms are involved in metal immobilization. Metal-P complex formation in the contaminated soil depends on the type of P amendment added, duration of soil-plant contact, type of plant species, and excretion of organic acids by the plant roots in the rhizosphere.