Development of a real-time PCR method for the differential detection and quantification of four solanaceae in GMO analysis: potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum)

The labeling of products containing genetically modified organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of beta-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification, and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intraspecies sequence variability), then using taxa involved in gene introgressions, and finally, using taxonomically close taxa (interspecies sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that beta-fructosidase can be used as an endogenous reference gene for GMO analysis.     

Citrantifidiene and citrantifidiol: bioactive metabolites produced by Trichoderma citrinoviride with potential antifeedant activity toward aphids

Two novel metabolites with potential antifeedant activity were isolated from cultures of the fungus Trichoderma citrinoviride strain ITEM 4484 grown in solid-state fermentation on sterile rice kernels. The producing strain was identified at species level by sequence analysis of the internal transcribed spacer regions ITS-1 and ITS-2 of the nuclear rDNA and a fragment of the translation elongation factor gene TEF-1alpha. Fractionation by column chromatography and TLC of the culture organic extract, followed by feeding preference tests on the aphid pest Schizaphis graminum (Rondani), allowed the purification of 5.8 and 8.9 mg/kg of culture of two bioactive metabolites, which were named citrantifidiene and citrantifidiol ( 1 and 2). Citrantifidiene and citrantifidiol, whose structures were determined by spectroscopic methods (NMR and MS) are a symmetrical disubstituted hexa-1,3-dienyl ester of acetic acid and a tetrasubstituted cyclohexane-1,3-diol, respectively. The pure metabolites influenced the feeding preference of S. graminum restraining individuals from feeding on wheat leaves dipped in 5% aqueous methanol solution containing 0.57 mg/mL of citrantifidiene or 0.91 mg/mL of citrantifidiol.     

Detection of full and limited amphidromous migratory dynamics of fish in Caribbean rivers

All native fishes living in the Caribbean island rivers are considered diadromous but few species have been validated as diadromous. In Guadeloupe (Lesser Antilles) as well as in the other tropical islands, the riverine habitats are subject to strong anthropogenic pressure due to resource modifications or dams. The management of fish biodiversity requires knowledge concerning their life history traits and behaviour that are still unknown for most species at a local scale and at the whole distribution area of the species. In a Guadeloupe river, we inferred the migratory patterns of four freshwater fishes common to Caribbean rivers by the analysis of otolith microchemistry. We found two species Eleotris perniger (Eleotridae) and Sicydium plumieri (Gobiidae) to be completely amphidromous with a marine larval phase and a freshwater adult phase. Agonostomus monticola (Mugilidae) was also found to be amphidromous in this system. The fourth species Gobiesox cephalus (Gobiesocidae) showed elemental ratios suggesting an estuarine larval phase and was defined as “limited amphidromous”. The identification of such life history traits, with limited dispersion abilities, argues for specific management measures in tropical island rivers at a basin scale for the conservation of biodiversity.     

Amblyomma variegatum in cattle in Marie Galante, French Antilles: prevalence, control measures, and infection by Ehrlichia ruminantium

We report Marie Galante as one of the Caribbean islands most heavily infested by the tropical bont tick (TBT) Amblyomma variegatum which is associated with two major diseases of ruminants: heartwater and dermatophilosis. In 2005, a survey was undertaken to assess the prevalence of TBT infestation in cattle, the prevalence of Ehrlichia ruminantium infection in TBTs, and the tick control measures implemented by livestock owners. A random sample of 195 cattle herds out of 1885 recorded on the island was investigated by thoroughly counting adult ticks on each animal and filling a questionnaire. A randomly collected sample of 136 TBTs was tested for infection by E. ruminantium by pCS20 nested PCR. Cattle herd prevalence (hp) was 73.8% for infestation by at least one TBT, 17.9% for infestation by at least one engorged female TBT, and 8.2% for clinical dermatophilosis. Cattle individual prevalence was 42.3% for infestation by at least one TBT, 6.6% for infestation by at least one engorged female TBT, and 2.2% for clinical dermatophilosis. The minimum, maximum and average numbers of TBTs per infested animal were, respectively 1, 108 and 11.5. Prevalence of TBT infection by E. ruminantium was 19.1%. No significant difference in herd prevalence was found among parishes or among ecological zones. For cattle owners treating against ticks (97.9% of all owners), all used aspersion of amitraz and herd prevalence was significantly different among those treating every 1-2-week (hp=69.6%, n=148), and less often than every 2-week (hp=88.6%, n=35) (P=0.031). Of the 42 herd subunits treated less than 4 days before the survey, 27 (64%) were infested with at least one TBT, and 6 (14%) with at least one engorged female TBT. These results indicate a high level of TBT infestation in Marie Galante, the inefficacy of tick treatments currently performed, and the need for an improved tick control strategy. Persisting high levels of infestation in Marie Galante threaten the success of on-going TBT eradication programs in the Caribbean because TBT can spread through migrating birds and trade of animals or of animal hides to other islands and potentially the American continent.     

Comparison of bronchoalveolar lavage cytospins and smears in dogs and cats

Differences in the cytological interpretation of bronchoalveolar lavage fluid (BALF) after cytospin preparation (CP) or manual smearing of pelleted cells preparation (MSP) were investigated in client-owned dogs and cats with inflammatory or infectious lower respiratory disease. Bronchoalveolar lavage fluid from healthy cats was also examined. With MSP, cell lysis was more frequently observed, and cellular distribution was more heterogeneous throughout the slide. When samples from healthy and diseased animals were considered together, a significantly greater percentage of neutrophils was seen on CP than on MSP slides (P<0.002). Cytospin preparations were considered of better quality in all individual comparisons. Cytospin preparation is advised in the evaluation of BALF with low total cell count. When only MSPs are evaluated, clinicians should be aware that differential neutrophil counts may underestimate the counts found on CP slides.     

Postoperative ultrasonographic appearance of uncomplicated enterotomy or enterectomy sites in dogs

The ultrasonographic findings in 20 dogs with 25 healing enterotomy and enterectomy sites resulting from the removal of foreign material or correction of intussusceptions are presented. In this prospective study, dogs had preoperative abdominal ultrasound examinations followed by sequential sonographic examinations on the first, third, sixth, and 10th days postenterotomy or enterectomy with an additional sonographic examination after 20 days postoperatively. Documented sonographic features included length and maximal intestinal wall thickness of the enterotomy or enterectomy sites, echogenicity of omental/mesenteric fat, amount of free gas and abdominal effusion, and gastrointestinal motility. Sonographically, 57% of enterotomies and 100% of enterectomies were visualized. Pneumoperitoneum, hyperechoic omental/mesenteric fat, and abdominal effusion did not appreciably hamper evaluation of the intestinal surgical site. Absent wall layering at the surgical site was noted in 96% of dogs at day 1 postoperatively. At the final sonographic examination, wall layering remained altered to absent in 100% of dogs and normal thickness was noted in only 20% of dogs. The median maximal wall thickness was 7 and 8 mm for enterotomies and enterectomies respectively, which occurred between days 1 and 3 and days 3 and 6 postoperatively. Effusion and increased echogenicity of omental/mesenteric fat localized to the surgical site were noted in 42% and 60% of dogs respectively at day 1 postoperatively, with resolution noted between days 3 and 10 postoperatively in 92% and 80% of dogs. Generalized abdominal effusion and pneumoperitoneum were seen in 100% of dogs immediately postoperatively and resolved in 80% by day 10.     

The role of Mycoplasma bovis in bovine respiratory disease outbreaks in veal calf feedlots

To assess the prevalence and relative importance of Mycoplasma bovis among the pathological agents implicated in bovine respiratory disease (BRD), we surveyed 135 veal calves from nine feedlots in eastern France during naturally occurring outbreaks of respiratory disease. Occurrence of respiratory pathogens, M. bovis, bovine viral diarrhoea (BVD) virus, bovine respiratory syncytial (BRS) virus and parainfluenza-3 (PI3) virus was investigated by seroconversion and isolation of bacteria and viruses from broncho-alveolar fluids. M. bovis and pathogenic respiratory bacteria were isolated in eight of the nine feedlots, and from 106 and 32, respectively, of the 135 tested animals. Seroconversion to PI3 virus occurred in four lots. BVD and BRS viruses were detected in eight and one lot, respectively. M. bovis was the most frequently isolated aetiologic agent in these BRD outbreaks, spreading early and widely throughout the affected units (60-100% rate of isolation and seroconversion). These results stress the importance of M. bovis in the BRD complex.     

Estimating symbiotic N2 fixation in Robinia pseudoacacia

Estimating symbiotic di‐nitrogen (N₂) fixation is challenging, especially when working with woody N₂ fixers in field trials. Fortunately, isotope methods based on ¹⁵N natural abundance or on ¹⁵N artificial enrichment (dilution method) make it possible to estimate the proportion of nitrogen derived from the atmosphere (Ndfa) in N₂‐fixing species. These methods have been extensively used in the field for herbaceous species, much less for tree species such as alder and acacia, and rarely for black locust (Robinia pseudoacacia). The objectives of this study were to characterize the fixation potential of black locust in a plantation by using the two ¹⁵N isotope methods in the field, and to document values of isotope fractionation occurring during N₂ fixation (the B value). B values were estimated both by growing trees on an N‐free medium in controlled conditions (B_lab) and by making Ndfa calculated with the natural abundance method converge with Ndfa calculated with the ¹⁵N dilution method in the field (B_field). The two methods gave consistent estimates of the B value. B values ranging between –1.4 and –3.2‰ were found, varying with the age of the plant material. Up to 76% of the N in the black locust trees came from the atmosphere, representing more than 45 kg N ha^?1 over five years and confirming that black locust may be well adapted to N‐poor soils.     

运用生物能量学模型预测草鱼生长、饲料需求和污染排放

为预测不同生长阶段草鱼生长性能、饲料需求量和污染排放量,提高草鱼投喂管理水平,本研究运用特定增长率(SGR)、日增长率(DGC)、日均增重(ADG)和热积温系数(TGC)等生长模型计算草鱼在不同生长阶段的生长速率,并通过计算定期采样中实际观测值和预测值最小残差平方和法选出最优生长模型。饲料需求模型通过估算鱼类消化能需求量决定,根据能量收支原理,通过计算鱼体储积能(RE)、基础代谢能(He E)、摄食热增能(Hi E)以及尿液和鳃的代谢能(UE+ZE),来估算草鱼的消化能,再根据所用饲料的消化能含量来确定草鱼对饲料的需求量。草鱼污染物排放主要采用营养物质平衡法计算。在模型验证时,以粗蛋白分别为33%、28%、23%的饲料投喂不同生长阶段的草鱼,将草鱼体质量和饲料系数(FCR)的模型预测值与实际观测值进行比较。结果显示,与其他生长模型(SGR、ADG、DGC)相比,调整后的TGC模型能更精确预测草鱼的生长情况;草鱼体质量和FCR预测值与观测值之间显著相关;每生产1 t鱼(体质量为0.5~2 500 g),其消化能需求量约为1.55×107 k J,消耗1 t饲料或生产1 t鱼所排放的总固态污染物分别为440和623 kg。研究表明,该复合性营养模型可以有效地估计实际养殖中草鱼生长、饲料需求量和污染物排放量,有望为草鱼差异化上市、节省饲料成本、减少饲料浪费以及养殖场的污染评价提供有效的预判工具。     

Phenotypic effects of antibiotic‐induced axenity and egg disinfection in early larval European seabass (Dicentrarchus labrax L.)

In the present study, the hypothesis that the difference in axenic conditions in the incubation and rearing environment of European seabass larvae induces size and shape effects on the specimens is tested. This difference is studied between xenic and axenic seabass larvae of DAH (day after hatching) 0, 5, 11 and 15. The axenic rearing protocol involves an egg disinfection with glutaraldehyde after the primary one with iodine in the hatchery, and the hypothesis that this secondary disinfection induces size and shape effects is also tested. In order to accomplish this, three egg and larvae treatments are included: “DA” (disinfected axenic), “DX” (disinfected xenic) and “NX” (nondisinfected xenic). Regarding the effect of antibiotic‐induced axenity, DA larvae exhibited larger bodies than both DX and NX on DAH 5 and 11. They also had a smaller yolk sac than DX at hatching, but consumed it slower. Towards the end of the experiment, DA larvae were thicker, but slightly more curved than DX and NX, which may be an abnormal shape, or a slightly more advanced ontogenetic stage. As far as egg disinfection, it had significant but very moderate shape effects on DAH 5 and 11, and disinfected larvae consumed their yolk sac faster. To the best of our knowledge, this study is one of the first to illustrate the subtle but significant size and shape effects caused by antibiotic‐induced axenity and secondary egg disinfection in the early larval stages, which suggest the existence of bacterial mechanisms that play a phenotypic role.