Antimicrobial activity of cuticle and outer eggshell protein extracts from three species of domestic birds

1. The eggshell cuticle is the proteinaceous outermost layer of the eggshell which regulates water exchange and protects against entry of micro-organisms. In this study, we investigated the hypothesis that the cuticle may also reduce microbial contamination by providing a chemical defence. 2. Outer eggshell and cuticle protein was extracted from domestic chicken (Gallus gallus), duck (Anas platyrhynchos) and goose (Anser anser) eggs by HCl and urea treatment, respectively. Antimicrobial activity of the extracts against Gram-positive and Gram-negative bacteria was evaluated. 3. C-type lysozyme, ovotransferrin and ovocalyxin-32 were identified in all extracts by Western blotting. All extracts from all species demonstrated lysozyme enzymatic activity. Immobilised c-type lysozyme retained some enzymatic activity. Protein extracts demonstrated activity against Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis suggesting the action of antimicrobial proteins in addition to lysozyme. 4. The results suggest that the antimicrobial outer eggshell and cuticle proteins present in a number of avian species may be a mechanism which enhances avian reproductive success.     

Technical note: detection of chicken, turkey, duck, and goose tissues in feedstuffs using species-specific polymerase chain reaction

PCR method was applied for the qualitative identification of chicken (Gallus gallus), turkey (Meleagris gallipavo), duck (Anas platyrhynchos x Cairina muschata), and goose (Anser anser) tissues in feed-stuffs, on an individual basis. The assay uses oligonucleotide primers that are specific for each avian species, targeting the 12S rRNA mitochondrial gene. The primers designed generated amplicons of 95, 122, 64, and 98 bp length for chicken, turkey, duck, and goose, respectively. The specificity of the primers was tested against 29 animal species including mammals, birds, and fish, as well as 8 plant species. Analysis of experimental feedstuffs demonstrated the detection of each target species in the range of 0.1 to 100%. The performance of this method was not affected by prolonged heat-treatment (up to 133 degrees C for 20 min at 300 kPa), and consequently, it could be very useful for the accurate identification of tissues from these 4 avian species in products submitted to denaturing technologies, for which other methods cannot be applied.     

The lack of binding ability of staphylococcal protein A and streptococcal protein G to egg yolk immunoglobulins of different fowl species (short communication)

The binding ability of staphylococcal protein A (SpA) and streptococcal protein G (SpG) to egg yolk antibodies of four fowl species (turkey, duck, moskovy duck and goose) was studied and compared with the binding ability to three serum antibodies from chicken, horse and cattle. SpA and SpG were not able to bind to any of the avian immunoglobulins.     

禽类全血DNA不同提取方法的比较

以4个不同禽类品种(金定鸭、长乐灰鹅、象洞鸡和闽南火鸡)为试验材料,采用4种不同方法从其全血中提取DNA,比较不同方法的DNA提取效率。结果表明:由于品种之间DNA存在差异,DNA的质量和产率也有显著差异,金定鸭全血DNA用树脂法效果最佳;长乐灰鹅和闽南火鸡全血DNA用酚-氯仿法效果最佳;而盐析法是提取象洞鸡全血DNA的最佳方法。     

Antibiotic Sensitivity Patterns among Indian Strains of Avian Pasteurella multocida

An investigation was carried out to study the antibiotic sensitivity of avian strains of Pasteurella multocida and to select an effective antimicrobial agent for control of avian pasteurellosis in India. A total of 123 strains of P. multocida recently isolated from different avian species (chicken, duck, turkey, quail, and goose), from different regions of India were subjected to antibiotic sensitivity tests using 20 different antibiotics. Absolute resistance was observed against sulfadiazine. The studies indicated that the strains were most sensitive to chloramphenicol (73.98%), followed by enrofloxacin (71.54%), lincomycin (64.23%), norfloxacin (61.79%) and doxycycline-HCl (56.91%). The majority of the strains were found to exhibit intermediate sensitivity. Chloramphenicol was selected and suggested for treatment. Antibiogram studies also revealed the emergence of multidrug-resistant strains of P. multocida among Indian poultry.     

Passage of duck hepatitis virus in cell cultures derived from avian embryos of different species

An egg-attenuated strain of duck hepatitis virus was successfully passaged through cell cultures of avian embryos derived from goose, turkey, guinea fowl, Japanese quail, pheasant and chicken. Two field strains of the virus were passaged in a more limited range of species.     

Comparative Staging of Embryo Development in Chicken,Turkey, Duck,Goose, Guinea Fowl,and Japanese Quail Assessed from Five Hours After Fertilization Through Seventy-Two Hours of Incubation

Normal tables of chicken embryo development are used to define specific stages of morphogenetic progression from the first cleavage divisions through hatching. Although established for the turkey and Pekin duck, the application of the normal tables of chicken embryo development to other birds of commercial and research importance needs be examined. Chicken, turkey, Japanese quail, and Pekin duck blastoderms from oviductal eggs showed differences in the rate of development that were inversely correlated with egg size. Oviposited eggs from these and additional species (goose, Muscovy and mule ducks, and Guinea fowl) were examined after 24 to 72 h of storage and at 6-h intervals up to 72 h of incubation. There was variation in the developmental stages of the blastoderm at the time of oviposition between and within the species and strains examined. Although it is recognized that the temporal rate of development will differ between different species and strains, the external features of any embryo in any given stage will be nearly identical.     

Sequence and phylogenetic analysis of interleukin (IL)-1beta-encoding genes of five avian species and structural and functional homology among these IL-1beta proteins

Interleukin (IL)-1beta-encoding regions of chicken, duck, goose, turkey and pigeon were cloned and sequenced. Each IL-1beta-encoding region of chicken, duck, goose and turkey is 804 nucleotides long and encodes IL-1beta protein that is 268 amino acids. Pigeon IL-1beta-encoding region is 810 nucleotides long and encodes IL-1beta protein that is 270 amino acids. Two one-nucleotide and one four-nucleotide insertions of pigeon IL-1beta-encoding region sequence were found, resulting in two amino acid insertions in pigeon IL-1beta. Pairwise sequence analysis showed that the sequence identities of IL-1beta-encoding genes ranged from 77% to 99%, which were also found for IL-1beta protein sequence identities, with an average level of both sequence identities of 89%. Phylogenetic analysis indicated that IL-1beta-encoding regions and the encoded proteins of chicken, duck, goose and turkey clustered together and evolved into a distinct phylogenetic lineage from that of pigeon which evolved into a second lineage. The results from the binding reaction of antiserum against each recombinant IL-1beta (r IL-1beta) protein to homologous or heterologous rIL-1beta, the enhancement levels of K60 mRNA expression in rIL-1beta-treated DF-1 cells or the reduction levels of K60 mRNA expression in DF-1 cells treated with rIL-1beta that was preincubated with homologous or heterologous antiserum showed that all five rIL-1beta were functional active and shared significantly structural and functional homology.     

Evaluation of a competitive ELISA for antibody detection against avian influenza virus

Active serologic surveillance is necessary to control the spread of the avian influenza virus (AIV). In this study, we evaluated a commercially-available cELISA in terms of its ability to detect AIV antibodies in the sera of 3,358 animals from twelve species. cELISA detected antibodies against reference H1- through H15-subtype AIV strains without cross reactivity. Furthermore, the cELISA was able to detect antibodies produced following a challenge of the AIV H9N2 subtype in chickens, or following vaccination of the AIV H9 or H5 subtypes in chickens, ducks and geese. Next, we tested the sensitivity and specificity of the cELISA with sera from twelve different animal species, and compared these results with those obtained by the hemagglutination-inhibition (HI) test, the "gold standard" in AIV sera surveillance, a second commercially-available cELISA (IZS ELISA), or the agar gel precipitation (AGP) test. Compared with the HI test, the sensitivities and specificities of cELISA were 95% and 96% in chicken, 86% and 88% in duck, 97% and 100% in turkey, 100% and 87% in goose, and 91% and 97% in swine, respectively. The sensitivities and specificities of the cELISA in this study were higher than those of IZS ELISA for the duck, turkey, goose, and grey partridge sera samples. The results of AGP test against duck and turkey sera also showed significant correlation with the results of cELISA (R-value >0.9). In terms of flock sensitivity, the cELISA correlated better with the HI test than with commercially-available indirect ELISAs, with 100% flock sensitivity.     

Molecular cloning, in vitro expression and bioactivity of goose B-cell activating factor

B-cell activating factor (BAFF), belonging to the TNF family, is critical for B cell survival and maturation. cDNA of goose BAFF (gBAFF) was amplified from goose spleen by RT-PCR. The open reading frame (ORF) of gBAFF encodes a protein of 288-amino acid. The gBAFF shows 98, 92, 44 and 55% amino acid sequence identity with duck (dBAFF), chicken (cBAFF), mouse (mBAFF) and human BAFF (hBAFF), respectively. RT-PCR results showed that gBAFF mRNA is expressed in thymus and more highly expressed in the bursa of Fabricius and spleen. Recombinant soluble gBAFF (gsBAFF) expressed in Escherichia coli has molecular weight of approximately 19kDa. In vitro, purified gsBAFF was able to promote bursa B cells survival/proliferation in goose, duck and chicken. Furthermore, recombinant dsBAFF and csBAFF have a positive effect on goose, duck and chicken bursa B cells survival/proliferation. These findings indicate that gBAFF plays an important role in the survival/proliferation of goose B cells and, owing to its high evolutionary conservation, functional cross-reactivity exists between chicken, duck and goose BAFF.     

Comparative investigation on the effect of T-2 mycotoxin on lipid peroxidation and antioxidant status in different poultry species

The effect of low dose T-2 toxin was investigated in chicken, duck and goose. The purpose of the present study was to investigate the effect of T-2 toxin on the lipid peroxidation and on the activity of glutathione redox system (amount of reduced and oxidised glutathione and the activity of glutathione peroxidase) in blood and liver. The treatment lasted days and two samples were taken, first at the time of lowest feed intake and second when the intake was the same as the control. It was found, that lipid per oxidation as detected by the amount of malondialdehyde increased in all of the species and tissues but the changes varied by species. The most sensitive species was goose followed by duck and chicken, and the most sensitive tissue was the liver followed by blood plasma and red blood cells.     

Cloning of PRL and VIP cDNAs of the Java sparrow (Padda oryzivora)

Complementary DNA (cDNA) of prolactin (PRL) and vasoactive intestinal polypeptide (VIP) of the Java sparrow were cloned and sequenced. The proximal region of the PRL promoter was also identified. Java sparrow PRL was found to have 88.3, 88.3, and 89.1% sequence identity at the cDNA level to PRL of chicken, turkey, and duck, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of chicken (91.4%), turkey (88.9%) and duck (92.0%) PRL. Based on the cDNA sequence and genomic structure of the chicken PRL gene, the proximal promoter was characterized. Sequence analysis of the proximal region of Java sparrow PRL promoter revealed a high degree of similarity to that of chicken, turkey and duck PRL promoters. Moreover, cDNA of prepro-VIP was also cloned and sequenced. Java sparrow prepro-VIP shows high similarity to chicken and turkey prepro-VIP. However, the region upstream of the 5' untranslated region of Java sparrow prepro-VIP did not show similarity to that of chicken. These results suggest that the mechanisms, which regulate expression of the VIP gene, may be different between precocial and altricial birds, but expression of the PRL gene may be widely conserved in avian species.     

北京鸭PRLR基因部分序列的克隆及分析

利用PCR方法克隆了北京鸭催乳素受体(PRLR)基因部分序列,该序列长1325bp。序列分析结果表明,该序列由第9外显子的一部分(57bp)、第9内含子(635bp)以及第10外显子的一部分(633bp)组成;在核苷酸水平上,北京鸭的该序列(除去内含子)与鹅(U07694)、鸡(D13154)、野鸽(DQ209271)、火鸡(L76587)的相似性分别为94.2%、88.7%、86.8%、86.1%;在氨基酸水平上,与鹅(ABA71353)、鸡(BAA02439)、野鸽(AAA20646)、火鸡(AAB01544)的相似性分别为93.5%、84.3%、80.8%、80.9%。     

家禽主要组织相容性复合体结构的研究进展

主要组织相容性复合体(MHC)存在于所有高等脊椎动物的基因组中,表现为高度多态和多基因的特性。MHC在免疫应答和病毒抵御上发挥着重要作用,不同家禽的MHC结构差异一定程度上可以解释其抗病性的差异。本文对近年来在鸡、鹌鹑、火鸡、鸭、鹅和其他几种禽类MHC结构研究方面的进展进行综述,对不同家禽MHC结构进行对比分析,有利于了解不同家禽品种间的遗传关系,促进家禽抗病育种的研究。     

鹅类新城疫病原研究

用鹅胚分别从扬州地区附近两地患病鹅群的病鹅肝、脾中分离到2株病毒,电镜观察病毒颗粒大小不等,形态不一,具有囊膜和纤突结构,病毒粒子大小100～300nm。两毒株能凝集鸡和人O型红细胞,并为鹅康复血清所抑制,对鹅、鸡及鹅胚、鸡胚、鸭胚都有致病力,但对鸭无致病力,人工感染健康鹅可复制出与自然病例一致的病状,病毒对鹅胚LD（50）为10(10.4）,MDT67．4h,鹅的LD（50）为10(7.5)。用鸡新城疫Ⅰ系苗对该鹅病有良好预防效果。根据病毒鉴定、临床表现及免疫效果将该病暂定为鹅类新城疫。     

Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism

Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different enzymes HhaI and HpaII produced 9 and 18 clusters respectively, whereas Single enzyme-AFLP recognized 32 patterns out of 72 strains typed. The study indicated that REA using HpaII is a simple and resource efficient method, however, further typing with more stringent and rapid method like Single enzyme-AFLP, could drastically enhance investigation in epidemiological studies of fowl cholera outbreaks.     

北京鸭β-catenin基因cDNA的克隆和生物信息学分析

试验探讨了北京鸭β-catenin基因的生物学功能,为进一步研究该基因对鸭皮肤毛囊的影响奠定基础。以北京鸭胚胎皮肤组织为材料,根据GenBank中发表的鸡、鹅等物种的β-catenin基因序列,设计并合成4对引物,采用RT-PCR方法,克隆北京鸭β-catenincDNA,并运用DNAMAN软件及在线工具对所得到的序列进行生物信息学分析。结果表明:北京鸭β-catenin基因cDNA长度为2996bp(Genbank登录号为FJ169885),包含由2346个碱基组成的编码区(CDs),有起始密码子ATG和终止密码子TAA,编码781个氨基酸;北京鸭β-catenin基因核苷酸序列与鸡、鹅、中华鳖、人和家猪相应区段(CDs)的同源性分别为95.06%、94.12%、90.11%、84.83%、84.31%;其氨基酸序列与鸡、鹅、中华鳖、猪、人的氨基酸同源性达99%以上,表明在进化关系上,β-catenin氨基酸序列有很高的保守性。     

Infections Investigation of ALV and REV in Guangxi Local Poultry Breeds and Sequence Analysis of gp85 Gene of ALV Isolates

In order to investigate the infection status of avian leukosis virus (ALV) and avian reticuloendotheliosis virus (REV) in the major local breeds of Qinzhou,Guangxi,totally 953 samples of egg white,cloaca swab and serum of Ma duck,Shitou goose,Tiejiao-Ma chicken,turkey and pigeon were collected from the representing flocks and detected by the commercial ELISA kits.ALV was isolated for the ALV p27 positive samples by culturing on DF-1 cells,and gp85 gene was sequenced.The results showed that the detections of ALV were negative in the samples except those of Tiejiao-Ma chicken,while REV antibody was found positive in Ma duck,Tiejiao-Ma chicken and turkey.The nucleotide sequences of gp85 gene of two isolates shared 94.5% identity with each other,and shared 86.9% to 94.9% with reference strains.The amino acid sequences of gp85 gene of two isolates shared 91.5% identity with each other,and shared 84.0% to 91.6% with reference strains.There were many variable sites in the hyper variable region hr1 and hr2,and the vr2 and vr3 variable regions were relatively conservative.Phylogenetic tree analysis showed that the two isolates shared the highest homology with SCAU11-XG strain.     

广西地方禽类品种ALV和REV感染情况的调查及ALV分离株gp85基因序列分析

为了解广西钦州地区主要地方品种商品禽类中禽白血病病毒(ALV)、禽网状内皮组织增殖症病毒(REV)的感染情况,本试验随机采集了广西钦州地区代表饲养场的麻鸭、狮头鹅、铁脚麻鸡、火鸡、鸽子共5个品种的蛋清、肛拭子及血清样品共953份,使用ELISA商品试剂盒进行检测;然后对部分ALV p27阳性的个体采集其血清样品接种DF-1细胞进行病毒分离并测定其gp85基因的序列。结果发现,除铁脚麻鸡外其他4个非鸡禽类品种的ALV检测均为阴性;除鸽子和狮头鹅外,麻鸭、铁脚麻鸡、火鸡均检测到REV抗体阳性;获得的两株铁脚麻鸡ALV分离株gp85基因之间核苷酸同源性为94.5%,与参考株之间核苷酸同源性为86.9%~94.9%;两株分离株gp85基因之间氨基酸同源性为91.5%,与参考株之间氨基酸同源性为84.0%~91.6%。高变区hr1和hr2区存在较多可变位点,可变区vr2和vr3相对较保守。系统进化树分析结果表明这两个毒株与参考株SCAU11-XG的亲缘关系最近。     

Immunoglobulin G Fc receptors of Mycoplasma synoviae

The objective of the study was to demonstrate and characterize IgG Fc receptors of Mycoplasma synoviae. Two IgG Fc receptors were recognized with molecular weights (MW) of 80,000 and 90,000 and isoelectric focusing points (pI) of 5.3 and 4.3, respectively. The activity of the IgG Fc receptors was eliminated by exposure to 0.1 unit of protease for 10 minutes. Mild reduction in activity was observed with trypsin between 100 to 1000 units for 10 minutes. The IgG Fc receptors were resistant to exposure to 60 C for 60 minutes and to 100 C for 20 minutes. The M. synoviae IgG Fc receptors were strongly reactive to affinity-purified Fc Fragment of chicken IgG; affinity-purified chicken IgG; and serum IgG of chicken, quail, pigeon, and turkey. A moderate reaction was detected to human affinity-purified IgG, and weak reactions were detected to affinity-purified IgG of cat, cow, dog, goat, guinea pig, horse, and rabbit. No reaction occurred with IgG of duck, goose, mouse, pig or rat.