佛山市食品中致泻大肠埃希菌菌型分布及毒力研究

目的 了解佛山市食品中致泻大肠埃希氏菌的菌型分布及毒力携带情况。方法采集佛山市十类食品362件中分离的17株致泻大肠埃希氏菌。按GB4789．6-94国家卫生标准检验方法进行血清分型；用ELISA检测耐热肠毒素(ST)和不耐热肠毒素(LT)；毒力测定采用小鼠致病力试验和采用irp2和irpB基因探针菌落原位杂交检测ES-IEC菌株毒力岛。结果 菌型分布在三类11个型别上，其中EPEC占41．17％(7／17)，EIEC为35．29％(6／17)。ESIEC为23．53％(4／17)；17株致泻大肠埃希氏菌在七类食品中均有存在。对小鼠致病力试验均为阳性；耐热肠毒素(ST)和不耐热肠毒素(LT)均为阴性，其中4株ESIEC中1株携带耶尔森氏菌强毒力岛(HPI)。结论佛山市食品中污染致泻大肠埃希氏菌普遍，对小鼠致病力与耐热和不耐热肠毒素无必然联系；有携带耶尔森氏菌HPI的ESIEC存在。应引起足够重视。     

两种DNA的PCR产物克隆入同一载体的构建策略

目的：以克隆小肠结肠炎耶尔森菌强毒力岛可变区首尾区域的双独立片段为例,探讨克隆入同一载体的基因数多于一个时可采取的构建策略。方法：在一个基因A正义引物与另一基因B负义引物的5′端分别设计与克隆载体相匹配的酶切位点,在 B基因正义引物与A基因负义引物的5′端设计相同的酶切位点。分别进行PCR扩增,再通过引物的5′端相同的酶切位点进行两PCR产物的酶切与连接,以连接产物为模板,应用A基因正义引物和B基因负义引物进行PCR扩增,将此次PCR产物经常规方法克隆入载体。     

Ontogeny of corticotropin-releasing factor and of hypothalamic-pituitary-interrenal axis responsiveness to stress in tilapia (Oreochromis mossambicus; Teleostei)

The ontogeny of the corticotropin-releasing factor (CRF) system and of the ability of the hypothalamic-pituitary-interrenal (HPI) axis to respond to stressors (capture or confinement), or to cortisol treatment was investigated in tilapia (Oreochromis mossambicus). In 2 days post hatching (dph) larvae, the first developmental stage used for immunohistochemistry, CRF-immunoreactivity (ir) was observed in the nucleus preopticus (npo), and in two hypothalamic nuclei (nlt and nrl). In this stage, CRF- and AVT-ir was found in the neural part of the pituitary, and endocrine cells in the pars distalis and pars intermedia contained POMC-derived peptides. In the ventral telencephalon, CRF-ir cells were first observed 5 dph, whereas projections from these cells into the anterior part of the latero-dorsal telencephalon (Dla) from 7 dph onwards. CRF, ACTH, alpha-MSH, and cortisol were quantified by radioimmunoassays in homogenates of the anterior-cranial region of the larvae containing brain, pituitary, and headkidneys. CRF contents increased from 43 +/- 3 to 1070 +/- 70 pg/larvae between 5 and 110 dph. Larvae of age 5, 12, 24, and 42 dph were captured sequentially from a group. All life stages were able to rapidly increase their cortisol content in response to this stressor (ANOVA: P < 0.001). Overall, the developmental stage affected cortisol content (ANOVA: P < 0.001), but developmental stage did not influence the cortisol reaction to stress (ANOVA: P > 0.162). Whole brain CRF content did not change during the 20 min stress period and the relationship between CRF-producing neurons and the initial HPI stress response in early life stages remains to be established. Cortisol feeding of 18 and 29 dph larvae for periods ranging from 2 to 24 days resulted in elevations of the CRF content (P < 0.003) in comparison to controls. In 18 dph larvae cortisol feeding abolished the cortisol response to capture stress as observed in control fed larvae (P < 0.008). We propose that cortisol induced upregulation of CRF takes place in the telencephalon and is restricted to a time period during larval development, characterised by the absence of glucocortoid receptor (GR) expression in the telencephalic Dm region in these larvae. Finally, the stress response to 24 h confinement was compared between saltwater adapted and freshwater adapted juveniles (age 77 dph). Confinement stress (24 h) affected cortisol and CRF content (ANOVA: P < 0.001, P < 0.008, respectively), but not ACTH content. Interactions were observed between salinity and confinement regarding cortisol and alpha-MSH contents (ANOVA: P < 0.02), but not regarding CRF and ACTH contents. The increase in cortisol levels induced by confinement was remarkably high in freshwater adapted larvae (five times higher than in saltwater adapted larvae). Regarding the cortisol response it is concluded that during and after the period of mouth breeding tilapia larvae respond to capture stress in a similar fashion (onset and height) as adults. Previously, we reported that the initial plasma cortisol response to capture stress in adult tilapia occurred independently from changes in plasma ACTH levels. The current finding that also brain CRF contents do not alter during the initial cortisol response in larvae further indicates that the initial cortisol response in this species may be regulated independently from CRF and ACTH.     

Corticotropin-releasing factor and neuropeptide Y mRNA levels are elevated in the preoptic area of socially subordinate rainbow trout

The objectives of this study were to characterize rainbow trout (Oncorhynchus mykiss) corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) cDNAs and to determine their mRNA levels in response to social stress. Standard cloning techniques were used to obtain cDNAs, sequences for trout NPY and two CRF isoforms. At the predicted amino acid level, our NPY sequence differs from the trout amino acid sequence reported by. A phylogenetic analysis suggests that the two CRF isoforms result from a gene duplication that occurred in a common ancestor of salmonids. A tissue distribution demonstrated that the mRNAs of both CRF isoforms are predominantly present in the preoptic area of the trout brain, whereas NPY mRNA is more abundant in the telencephalon. Pairs of sized-matched juvenile female trout were allowed to interact for 72 h and social ranks were assigned on the basis of behavioural observations. Mean plasma cortisol levels were 13-fold higher in subordinate than in dominant trout. As measured by ribonuclease protection assay, CRF1 and NPY mRNA levels were respectively 51 and 32% higher in the preoptic area of subordinate trout; in addition, CRF1 and NPY mRNA levels were positively correlated (R2=0.44). These results suggest that subordinate rainbow trout chronically maintain high levels of CRF mRNA during social stress and that NPY may be involved in the control of the stress axis in trout.