The neonatal Fc receptor (FcRn) is expressed in the bovine lung

In neonatal calves, maternal immunoglobulin (Ig) is transferred into respiratory secretion which contributes to protection against pathogens. The early predominance of IgG1 in respiratory tract secretions is progressively reduced in favor of IgA by age but in the lower, bronchoalveolar system secreted IgG remains the dominant secreted Ig even in adulthood. The trans-epithelial transport of secretory IgA into mucosal secretions is carried out by the polymeric Ig receptor. However, the mechanism by which IgG crosses epithelial cells to provide defense on mucosal surfaces is still unknown. In order to investigate the possibility that the neonatal Fc receptor, FcRn is involved in this transport we have first analyzed the localization of this receptor in the upper and lower respiratory tracts. Consistent with the in situ hybridization data, immunohistochemistry showed undetectable expression in the tracheal epithelial cells, relatively weak expression in epithelial cells of the bronchi, apparent staining those lining the bronchioli and randomly scattered signal over the alveolar tissue. The bovine FcRn may thus play a role in IgG transport across mucosal epithelial barriers as a trafficking receptor and ensure IgG predominance in the lower respiratory tract.     

Molecular cloning and identification of full-length cDNA encoding high affinity Fc receptor for bovine IgG (Fc gamma RI)

The receptor I for the Fc region of immunoglobulin G (Fc gamma RI) is a member of the Ig superfamily with a high affinity, and it mediates antibody-dependent cellular cytotoxicity and immune complex clearance. In this study, a cDNA encoding the bovine Fc gamma RI was cloned. The full-length cDNA sequence is 1050 bp long with a short 5'- and long 3'-untranslated end regions, which codes for 349 amino acids and contains a signal peptide, an extracellular region with three Ig-like domains, and transmembrane and intracytoplasmic domains. Five potential N-linked glycosylation sites are recognized in this sequence. Compared with the sequences of human and mouse Fc gamma RI, the homologies of nucleotide sequences are 80 and 69% and homologies of deduced amino acid sequences are 66 and 55%, respectively. It is shown that the sequences of the monomeric IgG binding domain in these three species of Fc gamma RI are highly conserved.     

牛干扰素-γ基因的RT-PCR扩增、序列分析及其在大肠杆菌中的表达

根据GenBank上发表的牛干扰素γ基因序列设计引物,从牛外周血淋巴细胞中提取基因组RNA,采用RTPCR技术,扩增出干扰素γ基因并进行序列分析。琼脂糖凝胶电泳显示牛干扰素γ扩增片段约为500bp。序列分析结果表明,牛干扰素γ基因序列全长517bp,开放阅读框架内501个核苷酸,共编码166个氨基酸,分子质量19.4ku,与参考序列完全一致。克隆序列经BamHⅠ/EcoRⅠ双酶切后连接到经同样双酶切的PBV220质粒中,转化入大肠杆菌DH5α中,筛选阳性克隆进行温度诱导表达,经SDSPAGE分析,在约19.4ku处有表达的蛋白带,表达产物经免疫荧光染色鉴定为阳性。     

Expression of the mouse Fc receptor B2 in bovine cells rescues the infectivity of conditionally neutralized bovine viral diarrhea virus

We examined the infectivity of bovine viral diarrhea virus (BVDV) particles opsonized with monoclonal antibodies on bovine cells expressing the murine Fcgamma receptor B2 (FcgammaRB2). Incubation of BVDV with each of five monoclonal antibodies (Mabs) to the envelope glycoprotein E2 led to efficient virus-neutralization, as evidenced by the failure to infect standard bovine testicle cells. In contrast, inoculation of four of these Mab-virus complexes onto transfectant bovine testicle cells expressing FcgammaRB2 resulted in a significant rescue of virus infectivity. Mab-virus complexes were 13.1, 7.37, 5.56 and 4.49 times more infectious for FcgammaR-expressing cells than for cells lacking FcgammaR. Because Mab-opsonized BVDV virion complexes uninfectious for standard cells may initiate productive infection in cells expressing the FcgammaR, the virion-Mab interaction should be described as a conditional neutralization. Interestingly, the infectivity of BVDV complexed with a specific virus neutralizing Mab (10f9) could not be rescued in FcgammaRB2-expressing cells. We postulate that attachment of antibody-virus complexes to FcR may only result in productive infection if the binding of antibody to virions does not interfere with post-attachment entry functions. Conditionally neutralized virions may play a role in the pathogenesis of any of the multiple diseases resulting from BVDV infections in cattle.     

牛IgG2Fc受体在COS-7细胞表面的稳定表达

将牛IgG2 Fc受（boFcy2R）编码区cDNA亚克隆到真核表达载体pcDNA3的巨细胞病毒启动子下游，构建了重组表达质粒pc3b02R；以重组质粒转染COS-7细胞，用玫瑰花环试验检测boFcy2R在转染细胞表面的表达，通过G418抗性筛选和连续克隆化，在转染细胞表面稳定表达了boFcy2R受体分子，转染细胞的玫瑰花环形成率达90％。最后获得稳定表达boFcy2R受体分子的COS-7转染细胞系，为受体的功能研究提供了良好的技术平台。     

Isolation of genes encoding bovine IgM, IgG, IgA and IgE chains

Bovine C mu, C gamma, C alpha and C epsilon genes were cloned in an EMBL4 recombinant phage library using rabbit immunoglobulin switch mu (Su) and human C gamma as probes. Restriction mapping and Southern blot analyses of these clones identified one clone which hybridized with rabbit C mu and JH probes. The HG and C mu regions were separated by 6 kb of DNA. One C alpha and one C epsilon gene were found on overlapping clones and were separated by approximately 15 kb of DNA. Southern blot analysis of germline DNA with a bovine C alpha associated probe (S alpha) indicated that the germline contains a single C alpha gene. Similar analyses with a bovine C epsilon probe indicated that the germline contains either one C epsilon gene with allelic restriction polymorphism or two C epsilon genes. Three C gamma genes were cloned and did not overlap with one another. Southern blot analyses of germline DNA with a bovine C gamma probe indicated that the germline contains a total of four C gamma genes. The genes cloned correspond to three of the four genes identified by Southern blot analysis. The orientation of each CH gene was assigned by hybridization with S mu or S gamma probes. The S gamma probe hybridized to DNA immediately adjacent to all three C genes; the S probe hybridized to DNA immediately adjacent to the C mu, C alpha and C epsilon genes. Unexpectedly, the S mu probe also hybridized with a segment of DNA approximately 7 kb downstream of the C mu gene. This may represent a switch region for C gamma.     

Polymorphism of 5'-region of the bovine growth hormone receptor gene

Genes coding for growth hormone (GH) and GH receptor (GHR) are candidates for quantitative trait markers in farm animals. This work describes a search for nucleotide sequence polymorphisms within the 5′‐region of the bovine GHR gene. Two new single nucleotide polymorphisms were found: restriction fragment length polymorphisms (RFLPs) at a Fnu4HI/TseI site (C/T transition at position ?1104), and at a Sau96I site (C/T transition at position ?262). The Fnu4HI/TseI polymorphic site is located within the 1.2‐kbp LINE‐1 retrotransposon upstream of the P1 promoter, while the Sau96I RFLP locates in the P1 promoter for exon 1A. The appearance of the Sau96I RFLP was studied in representatives of two bovine species, Bos taurus and Bos indicus. An absolute correlation was observed between Sau96I genotype and the insertion/deletion of LINE‐1.     

Surgery of the neonatal bovine digestive tract

Many disorders of the calf's gastrointestinal tract require surgical intervention if a successful outcome is to be obtained. The most common abnormalities in this category are abomasal volvulus, abomasal ulcers, small intestinal accidents, and atresia of the spiral colon. These can be differentiated by the age of the animal at presentation and a careful physical examination. Special considerations in neonatal gastrointestinal surgery include: ensuring adequate serum immunoglobulin status, rapid treatment of dehydration and hypoglycemia, and consideration of the inheritability of any corrected defects. Prompt attention to metabolic disturbances and correction of the abnormalities are essential for a successful outcome.     

Isolation of bovine herpesvirus-1 from vesicular lesions of the bovine udder

Bovine herpesvirus-1 was isolated from vesicular lesions on the udder and mammary papillae (teats) of a Charolais cow. Lesions on the animal consisted of papules and vesicles up to 10 mm in diameter. The virus was identified by fluorescent antibody and serum-neutralization tests.     

牛叠朊编码基因缺失突变体的构建及在大肠杆菌中的表达

本实验室已制备出多株针对牛叠朊的单克隆抗体,为充分鉴定这些单克隆抗体针对的抗原表位,通过基因克隆表达的策略获取牛叠朊基因缺失突变体的重组蛋白,以期为单克隆抗体的表位鉴定提供物质材料。经过对牛成熟叠朊编码基因及预测蛋白结构特征的分析,设计表达9个缺失突变体的引物。以PCR扩增出叠朊基因的缺失突变体,与pET-30a（＋）表达载体连接后转入E.coli DH5α中,经双酶切和测序鉴定后将重组质粒转入E.coliJM109和E.coli BL21表达宿主菌中,经IPTG诱导表达后,以SDS-PAGE、Western blot和ELISA检测表达产物的相对分子质量、相对表达量和反应原性。结果表明,成功构建了9个牛叠朊编码基因的缺失突变体,通过IPTG的诱导表达,其中有6个缺失突变体被大肠杆菌高效表达,并且具有较好的反应原性,这为其单克隆抗体表位的进一步鉴定奠定了物质基础。     

Costimulatory effects of complement receptor type 3 and Fc receptor for IgG (FcgammaR) on superoxide production and signal transduction in bovine neutrophils

The present study evaluated the costimulatory effects of complement receptor type 3 (CR3) and Fc receptor for IgG (FcgammaR) on superoxide production and intracellular signal transduction in bovine neutrophils. Stimulation with opsonized zymosan (OPZ) and heat-aggregated bovine IgG (Agg-IgG) resulted in much greater superoxide production and chemiluminescent (CL) responses in normal neutrophils compared with those stimulated with OPZ or Agg-IgG only. Superoxide production and CL response were closely associated with the stimulant-induced rise of the intracellular calcium ([Ca2+]i) concentration, amount of tyrosine phosphorylated 100 kDa protein, and activation of p38 mitogen-activated protein kinase (p38 MAPK). No costimulatory effect was found for these receptors on superoxide production in CR3-deficient neutrophils. Costimulation of CR3 and FcgammaR on bovine neutrophils leads to enhancement of superoxide production and their signaling pathways and appears to be associated with enhancement of neutrophil functions.     

Isolation of Pasteurellaceae from bovine abortions

A 2-year study was conducted to determine the incidence of Pasteurellaceae in abortion samples submitted for diagnostic evaluation. A total of 687 cases, including 623 with fetal tissues and/or stomach contents and 302 with placenta and/or uterine discharge, were evaluated. Pasteurellaceae were isolated on a nonselective medium from 9 (1.5%), 14 (2.8%), 13 (12.1%), and 42 (17.4%) of the fetal tissues, stomach contents, uterine discharges, and placentas, respectively. A total of 35 (19.9%) of 176 placental samples cultured on both a selective medium for Pasteurellaceae and a nonselective medium were positive for Pasteurellaceae. Fifteen (42.9%) of these isolates were detected only on the selective medium, whereas 5 (14.2%) were detected only on the nonselective medium and 15 (42.9%) grew on both media. Placentitis of different severity was evident in 13 (68.4%) of the 19 placentas from which Pasteurellaceae were isolated in the absence of other known abortifacient agents.     

Isolation of bovine complement factor H

A bovine serum protein, initially recognized by its inhibitory effect on the hemolytic activity of the bovine alternative pathway was isolated from fresh bovine serum by polyethylene glycol precipitation and chromatography on DEAE-Sephacel, CM-Sephadex A-50 and Sephadex G-200. The protein, a single chain polypeptide with an apparent molecular weight of 158,000, was identified as factor H, a regulatory protein of the alternative complement pathway. Functional characterization of this protein as factor H was based on the following properties: binding to C3b, inhibition of factor B binding to C3b, cofactor activity in the cleavage of C3b by factor I, inhibition of fluid phase alternative pathway C3 convertase (C3b.Bb) formation and activity, and species-specific inhibition of the alternative pathway mediated hemolysis of heterologous erythrocytes. A monospecific rabbit antiserum against bovine factor H failed to react with human serum factor H.     

Isolation of the fifth component of the bovine complement system

Bovine C5 has been isolated from fresh bovine serum by a five-step procedure: polyethylene glycol precipitation, sequential ion-exchange chromatography on DEAE-Sephacel and CM-Sephadex, hydroxylapatite chromatography, and affinity chromatography. The purified C5 was a protein of apparent molecular weight 202,000 +/- 9,000 composed of two chains: an alpha-chain of molecular weight 127,000 +/- 5,000 and a beta-chain of molecular weight 74,000 +/- 2,000. The alpha-chain was cleaved by Sepharose-CVF.Bb (a cobra venom factor (CVF)-induced C3/C5 alternative pathway convertase) in the absence of any C3 or C3b. The monocarboxylic acid form of K-76, a sesquiterpene compound isolated from the culture filtrates of Stachybotris complementi, inhibited the alternative pathway of bovine serum, and the inhibited hemolytic activity was restored, in a dose dependent manner, by bovine C5. This provided the basis for a C5 functional assay throughout the purification procedure. The purified C5 showed species specificity and was functionally distinct from bovine C3.