4个品种鸡Toll样受体4基因第2内含子的多态性分析

本研究采用PCR-SSCP和DNA测序的方法检测了4个品种鸡(仙居鸡、白耳鸡、清远麻鸡和安卡鸡)Toll样受体4(Toll-like receptor 4,TLR4)基因第2内含子的单核苷酸多态性。结果表明,4个品种鸡TLR4基因的第2内含子上均发现了G1894C的突变,共产生了AA、AB和BB 3种基因型,4个品种鸡该位点的多态信息含量(PIC)分别为0.570、0.597、0.583和0.588,均属于高度多态性,且处于Hardy-Weinberg平衡状态(P>0.05);4个品种鸡G1894C多态位点的基因型在感染鸡白痢沙门氏菌阳性组和阴性组间的分布差异均不显著(P>0.05);G1894C多态位点在仙居鸡与白耳鸡2个品种间基因型分布差异极显著(P<0.01)。     

合成绵羊抗菌肽NK-Lysin抗菌活性及其对雏鸡沙门氏菌攻毒的治疗效果研究

为了研究绵羊抗菌肽NK-Lysin主要活性区的生物学功能,试验设计并合成了3对功能区多肽片段,并利用径向扩散试验和最小抑菌浓度对其抗菌活性进行检测,分析了合成多肽对鸡血红细胞的毒性作用,筛选出抗菌效果最好的多肽对沙门氏菌攻毒的雏鸡进行治疗,检测其治疗效果。结果表明,合成多肽对大肠杆菌、沙门氏菌具有抑制活性,多肽片段长度、多肽C-端有无酰胺化及多肽内的二硫键是否成环对多肽的抗菌活性均有影响;筛选出的2个多肽在治疗雏鸡沙门氏菌攻毒的过程中能够明显降低死亡率,对雏鸡心脏、肝脏、肾脏的病理损伤也明显小于对照组。本研究结果为绵羊抗菌肽NK-Lysin作为候选抗菌药物的开发奠定了基础。     

Antimicrobial Activity of Synthesized Sheep NK-Lysin Peptides and Its Treatment for Chicken Salmonella Challenge

In order to study the biological function of the main active region of sheep antimicrobial peptide NK-Lysin, three pairs of functional domain peptide fragments were designed and synthesized the antibacterial activity was detected by radial diffusion test and minimal inhibitory concentration. We analyzed the toxic effects of chicken red blood cells and screen the best peptides for treatment chicken challenged by Salmonella pullorum in this study. The rusults showed that synthesized peptides were inhibitory to Escherichia coli and Salmonella pullorum. Fragment length of peptides, C-terminal amidation and peptide inner loop were essential to antibacterial activity. Two of synthesized peptides were used to treat chichen challenged by Salmonella pullorum which obviously decreased the mortality of chicken. The pathological damage of heart,liver,kidney were less than that of control group. This study laid a foundation for the development of the sheep NK-Lysin peptides as candidate antimicrobial agents.     

The small intestine of the adult New Hampshire chicken: an immunohistochemical study

The presence and distribution of glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide (GIP), gastric-releasing peptide (GRP) and glucagon immunoreactivity were studied in the small intestine of the New Hampshire chicken using immunohistochemistry. This is the first report of the presence of GIP-immunoreactive (ir) cells in avian small intestine. GIP, GRP and glucagon immunoreactivity was localized in the epithelium of the villi and crypts of the duodenum, jejunum and ileum. In particular, both in the duodenum and in the jejunum immunoreactive endocrine cells to GIP, GRP and glucagon were observed. In the ileum, we noticed GIP-ir and glucagon-ir cells. GRP-ir was found in nerve fibres of all three segments of the small intestine. The distribution of these bioactive agents in the intestinal tract of the chicken suggests that GIP and glucagon may play a role in the enteropancreatic axis in which intestinal peptides modulate pancreas secretion.     

Expression of chicken LEAP-2 in the reproductive organs and embryos and in response to <Emphasis Type="Italic">Salmonella enterica</Emphasis> infection

In recent years host antimicrobial peptides and proteins have been recognised as key mediators of the innate immune response in many vertebrate species, providing the first line of defense against potential pathogens. In chickens a number of cationic antimicrobial peptides have been recently identified. However, although these peptides have been studied extensively in the avian gastrointestinal tract, little is known about their function in the chicken reproductive organs and embryos. Chicken Liver Expressed Antimicrobial Peptide-2 (cLEAP-2) has been previously reported to function in protecting birds against microbial attack. The aim of this study was to investigate the expression of cLEAP-2 gene in the chicken reproductive organs, as well as in chicken embryos during embryonic development, and to determine whether cLEAP-2 expression in the chicken reproductive organs was constitutive or induced as a response to Salmonella enteritidis infection. RNA was extracted from ovary, oviduct, testis and epididymis of sexually mature healthy and Salmonella infected birds, as well as from chicken embryos until day ten of embryonic development. Expression analysis data revealed that cLEAP-2 was expressed in the chicken ovary, testis and epididymis as well as in embryos during early embryonic development. Quantitative real-time PCR analysis revealed that cLEAP-2 expression was constitutive in the chicken epididymis, but was significantly up regulated in the chicken gonads, following Salmonella infection. In addition, expression of cLEAP-2 during chicken embryogenesis appeared to be developmentally regulated. These data provide evidence to suggest a key role of cLEAP-2 in the protection of the chicken reproductive organs and the developing embryos from Salmonella colonization.     

兽用鸡源和猪源转移因子与免疫活性相关的多肽分析

为探明兽用鸡脾和猪脾转移因子(Transfer factor,TF)与免疫活性相关的多肽种类及其氨基酸组成,采用液相色谱-质谱联用法(LC-MS)对其进行多肽测序,并与蛋白质比对。结果发现,由鸡脾和猪脾制备的TF分别检测到免疫相关多肽45条和102条,多肽的氨基酸数量为5～24个,分子量为599.2784～2507.8258 Da;这些多肽对应于免疫活性蛋白质64种,以细胞免疫和先天免疫活性为主。该研究揭示了兽用鸡脾和猪脾TF的免疫活性多肽的组成及其氨基酸序列,有助于进一步研究其有效成分和作用机理。     

Serotonin-Containing Cells in the Horse Gastrointestinal Tract

The presence and distribution of serotonin-containing cells in the gastroenteric tract of horses have been investigated. The enterochromaffin (EC) cells have been identified using immunostaining procedures at both light and electron microscopic level. The EC cells were very numerous in the pyloric gland region, were only few in the duodenum but were absolutely lacking from the more distal portions of the intestine.     

Dissemination and tracking of Salmonella spp. in integrated broiler operation

Controlling Salmonella in integrated broiler operation is complicated because there are numerous potential sources of Salmonella contamination, including chicks, feed, rodents, wild poultry operations, and the processing plant. The objective of this study was to investigate the distribution of Salmonella through all phases of two integrated broiler operations and to determine the key areas related to the control of all known sources of infection. Two different Salmonella serotypes were observed at integrated broiler chicken company A. S. enteritidis, the predominant company A isolate, was consistently found in the breeder farm, hatcheries, broiler farms, and chicken slaughterhouse. At company B, a total of six different serotypes, S. heidelberg, S. senftenberg, S. enteritidis, S. blockley, S. gallinarum, and S. virchow, were detected. Although S. heidelberg was not found in the broiler farms, it was consistently found in the breeder farm, hatcheries, and chicken slaughterhouse. In addition, S. enteritidis was found in the hatcheries, broiler farm, and chicken slaughterhouse. In order to obtain the genetic clonality, 22 S. enteritidis isolates were digested with XbaI and analyzed by pulsed-field gel electrohporesis (PFGE). A difference in the PFGE pattern was found to be related to the origin of the integrated broiler operation. These data support the critical need to control Salmonella in breeder farms and hatcheries, and demonstrate important points related to the control of infection in large-scale poultry operations of Korea.     

超高效液相色谱串联四级杆/飞行时间质谱仪鉴定喹赛多在鸡体内代谢研究

采用超高效液相色谱串联四级杆/飞行时间质谱仪(UPLC-Q-TOF MS)识别和鉴定了喹赛多在鸡体内的代谢产物,并讨论了喹赛多在鸡体内可能的代谢途径。按800 mg/kg体重的剂量给鸡单次灌服喹赛多,采集试验前后鸡的粪便,经简单前处理后,运用UPLC-Q-TOF MS进行分析,采集的数据经Metabolynx^xs软件处理后,共鉴定出15种喹赛多的代谢产物。试验结果表明,喹赛多在鸡体内发生广泛的代谢。     

Determination of angiotensin-I converting enzyme inhibitory peptides in chicken leg bone protein hydrolysate with alcalase

This study aims to identify peptides with angiotensin-I converting enzyme (ACE) inhibitory activity in hydrolysate from chicken leg bone protein hydrolyzed with alcalase for 4 h (A4H). The hydrolysate has demonstrated potent in vitro ACE inhibitory activity, and has been shown to attenuate the development of hypertension and cardiovascular hypertrophy in spontaneously hypertensive rats (SHR). A4H is competitive for ACE and was separated using high-performance liquid chromatography (HPLC) with a gel filtration column (Superdex Peptide HR 10/30). The results show that A4H is a mixed non-competitive inhibitor. Eighteen fractions were detected after separation of A4H, and most of them showed ACE inhibitory activity. Five fractions with strong ACE inhibitory activities (above 50%) were labeled from A to E. In addition, there were 10 peptides, consisting of 5–10 amino acid residues that were identified from fraction D that exhibited the strongest ACE inhibitory activity. Three of the identified peptides corresponded to peptides derived from collagen type I and chicken muscular protein. It is revealed that A4H has several peptides that possess ACE inhibitory activities.     

Epitope mapping of Campylobacter jejuni flagellar capping protein (FliD) by chicken (Gallus gallus domesticus) sera

Campylobacter jejuni, a Gram-negative rod, is a zoonotic pathogen associated with human acute bacterial gastroenteritis worldwide. The flagellum, composed of more than 35 proteins, is responsible for colonization of C. jejuni in the host gastrointestinal tract as well as inducing protective antibodies against the homologous serotype. In our previous study, we demonstrated that the flagellar capping protein (FliD) is an immunodominant protein that reacted strongly to sera from field chickens. In this communication, we mapped linear immunoreactive epitopes on FliD using a set of 158 synthetic peptides of 15-mer overlapping with 11 amino acid residues on peptide microarrays with sera from field chickens. The results from peptide microarrays showed (1) no cross-reactivity of the immobilized peptides with the secondary anti-chicken antibody in the control incubation, and (2) heterogeneous patterns of sera reacting to the immobilized peptides. The peptides that reacted to more than three chicken sera and had higher averages of fluorescence units were selected for further validation by the peptide ELISA. The results showed peptides 24, 91 and 92 had relatively high reactivity and less variation among 64 individual serum samples, indicating these peptides represented the shared immunodominant epitopes on the C. jejuni FliD protein. These peptides were also recognized by sera from chickens immunized with the purified recombinant FliD protein. The findings of the specific shared linear immunodominant epitopes on FliD in this study provide a rationale for further evaluation to determine their utility as epitope vaccines covering multiple serotypes for chicken immunization, and subsequently, for providing safer poultry products for human consumption.     

Identification of an antihypertensive peptide derived from chicken bone extract

A novel angiotensin‐converting enzyme (ACE) inhibitory peptide was isolated and purified from chicken bone extract by enzymatic digestion. The peptide was defined as an ACE inhibitor, and it demonstrated antihypertensive activity following oral administration to spontaneously hypertensive rats (SHRs). The results of this study suggest that peptides derived from an extract of chicken bones, administered orally, have the ability to reduce the blood pressure of SHRs significantly over a short period of time (3 h). Moreover, the blood pressure then remains low for 3 h. This peptide derived from chicken bones may therefore have great value as a short‐term remedy for chronic conditions such as high blood pressure. The amino acid sequence of the peptide was YYRA (Tyr‐Tyr‐Arg‐Ala), which was the origin of the Ig heavy chain V region (27–30 position). The IC₅₀ value of its synthetic peptide was 33.9 μg/mL. We suggest that the ACE inhibitory and antihypertensive peptides derived from chicken bone extract may contribute to develop physiologically functional foods or improve food functionality.     

Th1/Th2 polarization by viral and helminth infection in birds

Mammals developed an immune system able to functionally polarize into so-called type 1 or type 2 immune pathways, to resolve infections with intracellular and extracellular pathogens, respectively. In the well-studied avian immune system of the chicken, however, no evidence for polarized immunity could be found, as yet. To investigate whether these two major arms of mammalian immunity, regulated by a T helper (Th)1/Th2 cytokine balance, evolved similarly in birds, chickens were exposed to a prevalent intracellular (viral) or extracellular (helminth) infection. By using semi-quantitative RT-PCR analysis we provide evidence that polarization of Th1/Th2 type immunity extends beyond mammalian species, and, therefore, has been evolutionary conserved for more than 300 million years, when the lineages of mammalian and avian vertebrates are assumed to have segregated.     

Intracerebroventricular administration of chicken glucagon‐like peptide‐2 potently suppresses food intake in chicks

Glucagon‐related peptides, such as glucagon‐like peptide (GLP)‐1, GLP‐2 and oxyntomodulin (OXM), are processed from an identical precursor proglucagon. In mammals, all of these peptides are suggested to be involved in the central regulation of food intake. We previously showed that intracerebroventricular administration of chicken OXM and GLP‐1 significantly suppressed food intake in chicks. Here, we show that central administration of chicken GLP‐2 potently suppresses food intake in chicks. Male 8‐day‐old chicks (Gallus gallus domesticus) were used in all experiments. Intracerebroventricular administration of chicken GLP‐2 significantly suppressed food intake in chicks. Plasma glucose concentration was significantly decreased by chicken GLP‐2, whereas plasma nonesterified fatty acid concentration was significantly increased. Intracerebroventricular administration of chicken GLP‐2 did not affect plasma corticosterone concentration. In addition, the anorexigenic effect of GLP‐2 was not reversed by the corticotropin‐releasing factor (CRF) receptor antagonist α‐helical CRF, suggesting that CRF is not a downstream mediator of the anorexigenic pathway of GLP‐2 in chicks. Intracerebroventricular administration of an equimolar amount of GLP‐1 and GLP‐2, but not OXM, significantly suppressed food intake in both broiler and layer chicks. All our findings suggest that GLP‐2 functions as a potent anorexigenic peptide in the brain, as well as GLP‐1, in chicks.     

Chicken RFamide-related peptide (GnIH) and two distinct receptor subtypes: identification, molecular characterization, and evolutionary considerations

RFamide-related peptides (RFRPs) regulate the release of various pituitary hormones in vertebrates. It is completely unknown how the functions of RFRPs vary among animal classes and whether vertebrate RFRPs are orthologous to each other and belong to the same peptide family. This report concerns identification of avian RFRP (gonadotropin-inhibitory hormone, GnIH) from the chicken. Chromosome-wide synteny conservation demonstrated the orthologous relationships among vertebrate RFRPs. The consensus motif for RFRP was modified to Pro-Xaa-Arg-Phe-NH2. We also describe the first identification of two distinct types of receptors for non-mammalian RFRP (RFRPR and NPFFR) from the chicken. Amino acid comparison revealed substantial differences in both termini of receptors among classes of vertebrates. The 5'-flanking regions of chicken RFRPR and NPFFR suggested their expressions in the pituitary gland, and this was confirmed by the RT-PCR analysis. Localizations of both chicken RFRP and its receptors were distinct from those of mammals. These results indicated that avian RFRP, unlike the mammalian one, directly acts on the pituitary gland via receptors to regulate gonadotropin release. It was also suggested that functional differences, especially between avian and mammalian RFRPs, are substantially due to divergences in the structures and expression sites of their receptors.     

Reconstruction of a chicken BF2 protein complex and identification of binding nonamer peptides derived from avian influenza virus hemagglutinin

In order to reconstruct the system for identification of short antigenic peptides, the chicken BF2 gene of Chinese Sanhuang (SH) chicken line was linked to the beta(2)m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich peptide, by splicing overlap extension PCR (SOE-PCR). The MBP-BF2-(G4S)3-beta(2)m fusion protein was expressed and purified in a pMAL-p2X/E. coli TB1 system. The purified MBP-BF2-(G4S)3-beta(2)m protein was cleaved by Factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the BF2-(G4S)3-beta(2)m protein was determined by circular dichroism (CD). In addition, the refolded BF2-(G4S)3-beta2m protein was used to bind three predicted nonameric peptides derived from the hemagglutinins of the avian influenza virus (AIV) H5N1 and H9N2 subtypes. The BF2-(G4S)3-beta2m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and tandem mass spectrometry analysis, respectively. The results indicate that the secondary structures and predicted three-dimensional crystal structure of BF2-(G4S)3-beta(2)m are similar to those of the monomers of chicken BF2 and beta(2)m. The BF2-(G4S)3-beta(2)m protein could bind two of the three predicted nonamer peptides derived from AIV hemagglutinin. The experimental system demonstrated that the reconstructed BF2-(G4S)3-beta(2)m protein complex can be used to identify nonamer peptides, including T-cell epitopes in chicken.     

肌细胞生成素基因多态性与泸宁鸡生长性状的关联研究

本研究旨在探讨肌细胞生成素(myogenin,MyoG)基因多态性与泸宁鸡生长性状的关联性.采用PCR-SSCP和DNA测序技术,对泸宁鸡MyoG基因5'调控区的多态性进行检测.结果表明,泸宁鸡MyoG基因5'调控区存在4个多态位点(A153G、A267G、A446G和C612T).在153 bp处存在A/G突变,形成3种基因型(AA、AG和GG),其中AA基因型肝脏重显著高于AG、GG基因型(P <0.05).在446 bp处存在的A/G突变,形成3种基因型(AA、AG和GG),该位点GG基因型屠宰率显著高于AA、AG基因型(P <0.05),AG、GG基因型胸肌率显著高于AA基因型(P <0.05).以上结果显示,MyoG基因5'调控区的多态性与泸宁鸡肝脏重、屠宰率、胸肌率有一定的关联性,可作为泸宁鸡相应生长性状的候选基因.     

Distribution of orexin B and its relationship with GnRH in the pig hypothalamus

Increasing evidence suggests that orexins--hypothalamic neuropeptides--act as neurotransmitters or neuromediators in the brain, regulating autonomic and neuroendocrine functions. Orexins are closely associated with gonadotropin-releasing hormone (GnRH) neurons in the preoptic area and alter luteinizing hormone (LH) release, suggesting that they regulate reproduction. Here, we investigated the distribution of orexin B (immunohistochemical technique) and the relationship between orexin B and GnRH containing fibres and neurons in the pig hypothalamus using double immunofluorescence and laser-scanning confocal microscopy. Orexin B immunoreactive neurons were mainly localized in the perifornical area (PeF), dorsomedial hypothalamic nucleus (DMH), zona incerta (ZI) and the posterior hypothalamic area (PH), with a sparser distribution in the preoptic and anterior hypothalamic area. Immunoreactive fibres were distributed throughout the central nervous system. Approximately 30% GnRH neurons were in close contact with orexin B immunoreactive fibres, among these approximately 6% of GnRH neurons co-localized with orexin B perikarya in the region between the caudal preoptic area and the anterior hypothalamic area. Orexin B may regulate reproduction by altering LH release in the hypothalamus.     

The distribution of Salmonella serovars in chicken and humans in Lithuania

We studied serovars of Salmonella strains isolated from chicken and humans in Lithuania over the period from 2000 to 2004. Salmonella strains were isolated and identified according to the techniques recommended by International Organisation for Standardization (Microbiology of Food and Animal Feeding Stuff--Horizontal Method for the Detection of Salmonella, 1998, ISO, Geneva). The per cent of infected flocks with Salmonella in separate years was between 1.01% and 3.2% during the period of investigation. The contamination rate of broiler legs and breasts was higher (2.36% and 4.25%) than that of wings (0.82%). Eighteen serovars of Salmonella were identified from the total 300 isolated samples. The most prevalent serovars in chicken were Salmonella Enteritidis, Salmonella Infantis and Salmonella Typhimurium. Other serovars such as Salmonella Montevideo, Salmonella Djugu, Salmonella Isangi, Salmonella Bovismorbificans, Salmonella Mbankada, Salmonella Hadar were detected only in one to two samples. In general, similar serovars of Salmonella were found in humans and chicken (S. Enteritidis and S. Typhimurium), although distinct serovars were found only in humans or only in chicken. Analysis of the distribution of Salmonella serovars in humans during the seasons of the year indicated that the highest incidence of Salmonella was in Summer and in the beginning of Autumn. Analysis of the distribution of serovars during the study period indicated that there is a shift over time in both humans and chicken.