Survival of Corynebacterium renale, Corynebacterium pilosum and Corynebacterium cystitidis in soil

Survival of the causative agents of bovine pyelonephritis, Corynebacterium renale, C. pilosum and C. cystitidis, was examined at 30 degrees C in autoclaved soil. In the soil from a paddock, C. renale and C. cystitidis survived for 56 and 63 days, respectively, and C. pilosum for a longer period of at least 210 days. In soil from a pasture, sand from an athletic field and sea sand, the survival of these bacteria was of shorter duration.     

Phase variation of pili of Corynebacterium pilosum.

Whether or not (?) phase variation occurs in the pili of Corynebacterium pilosum was biologically examined using the colony enzyme-linked immunosorbent assay (ELISA) blot test with anti-pili immune serum. From the densely piliated clone (35P+) of C. pilosum 35, non-piliated variants were isolated at a frequency of 2.45 x 10(-3). From one of the non-piliated variants (designated as P(-11)/35P+), a piliated variant was isolated at a frequency of 4.68 x 10(-4), one log less frequently than the non-piliated variant. From this piliated variant as designated (P+4/P(-11)/35P+), a non-piliated variant was isolated at a frequency of 3.86 x 10(-3). C. pilosum was thus alternated between piliated and non-piliated at a fairly high frequency, suggesting that the pili may undergo phase variation. This is the first finding of phase variation of the pili of Gram-positive bacteria.     

Adhesion of Corynebacterium renale and Corynebacterium pilosum to epithelial cells of bovine vulva

Corynebacterium renale and C pilosum adhered effectively to the epithelial cells of the bovine vulva; the numbers of these organisms that adhered to the vulval epithelial cells were 50 and 30/cell, respectively, which were several times as many as those that adhered to the uroepithelial cells. Of the epithelial cells of the vulva, cornified cells lacking nuclei bound more bacteria than did those with indistinct nuclei, indicating that adhesion of bacteria was most effective to the most aged cells. The marked adhesion of C renale and C pilosum to the epithelial cells of the vulva may indicate that the vulva is an important portal of entry of these bacteria.     

Comparison of surface hydrophobicity of piliated and non-piliated clones of Corynebacterium renale and Corynebacterium pilosum

Piliated (P+) and non-piliated (P-) clones of Corynebacterium renale and C. pilosum were similar in hydrophobicity as measured by hydrophobic interaction chromatography, bacterial adherence to hydrocarbons and the salt aggregation test. Therefore, the previously reported adherence of P+ clone to various cells, which is more effective than that of P- clone, may be uncorrelated with the degree of hydrophobicity of both clones of these bacteria. Hydrophobicity of P+ and P- clones was found to be high when measured by hydrophobic interaction chromatography and bacterial adherence to hydrocarbons, but low when measured by the salt aggregation test.     

Adhesion of Corynebacterium pilosum by pili to epithelial cells of bovine vulva

Piliated (P+) and nonpiliated (P-) clones of Corynebacterium pilosum were selected, and their adhesion to the epithelial cells of the bovine vulva and vaginal vestibule was examined. The number of P+ bacteria of C pilosum that adhered to vulval epithelial cells was greater than that of P- bacteria. The adhesion of P+ bacteria, but not P- bacteria, to the epithelial cells was inhibited by the antipilus antiserum; therefore, the adhesion of C pilosum to the epithelial cells of the vulva was primarily dependent on the pili. The number of C pilosum that adhered to the epithelial cells of the bovine vulva and of the vaginal vestibule increased by decreasing the pH.     

Attachment of Corynebacterium renale to tissue culture cells by the pili.

One or more cells of Corynebacterium renale strains (serologic types, I, II and III), which possessed numerous pili, frequently were attached to BHK-21 cells, primary dog kidney cells, and primary rabbit kidney cells. The percentage of the cultured cells to which C renal cells were attached was about 70%. The percentage was less with cells of C renale possessing fewer pili, around 30%. After C renale was treated with the homologous anti-pili serum, the percentage of BHK-21 cells to which bacterial cells were attached was even less (22%). In electron micrographs, the pili of C renale were observed to attach themselves to the membranes of BHK-21 cells. The adhesive property of the pili of C renale to tissue culture cells was thus demonstrated.     

Cloning and expression of a pili gene of Corynebacterium renale in Escherichia coli

A plasmid gene library of Corynebacterium renale piliated strain No. 109P+ was prepared in Escherichia coli in order to study the chemical structure of the pili of C. renale. Of 3,000 recombinant clones tested, 5 reacted with anti-pili anti-serum. The gene products of these clones reacted with anti-pili monoclonal antibodies 8/4, 5/2 and B20/3 but lacked the reactivity with 13/4. SDS-PAGE analysis revealed that the expressed protein had a molecular mass of 48 kilodalton and deletion analysis showed that the encoding region for this protein was localized within a 1.4 kilobase gene including a promoter sequence. Immunoelectron microscopy showed that mouse antibodies raised to the expressed protein bound to the entire surface of the pili of C. renale. These results indicate that the cloned gene encodes a major structural protein of C. renale pili.     

Isolation and characterization of monoclonal antibodies against structural proteins of infectious laryngotracheitis virus

Thirteen infectious laryngotracheitis virus (ILTV)-specific monoclonal antibodies (MAbs) were isolated after immunization of mice with purified infectious laryngotracheitis virions. On the basis of their reactions in western blot analyses of ILTV-infected cells, the MAbs were assigned to five different virus proteins or protein groups. Two of the viral target proteins could be identified after transient expression of cloned ILTV genes in eucaryotic cells. The MAbs of group II detected a 60-kD protein that was shown to be the ILTV homologue of herpes simplex virus type 1 (HSV-1) glycoprotein (g)C. The MAbs of group I reacted with the positional homologue of HSV-1 gJ, which is encoded by the open reading frame (ORF) 5 gene within the unique short genome region of ILTV. The ORF 5 gene product of ILTV was previously described as a 60-kD glycoprotein (gp60), whereas multiple protein bands with apparent molecular masses of 85, 115, 160, and 200 kD were identified in the present study. Immunoelectron microscopy revealed that both gC and gJ of ILTV are localized in the envelope of virus particles, whereas the 15-kD protein detected by the MAbs of group III presumably represents a tegument component. Immunofluorescence analyses of infected cells demonstrated that the epitopes of the gC- and gJ-specific MAbs are conserved in all tested ILTV isolates originating from different parts of the world and that these MAbs are also suitable for in situ antigen detection in tissues of ILTV-infected chickens. The remaining ILTV-specific MAbs recognized viral proteins of 22 kD (group IV) and 38 kD (group V) that were not further characterized up to now.     

Adhesion of Corynebacterium renale and Corynebacterium pilosum to the epithelial cells of various parts of the bovine urinary tract from the renal pelvis to vulva

The various parts of the bovine urinary tract, the renal pelvis, the ureter, the urinary bladder, the urethra, the vaginal vestibule and the vulva, were examined for the capacity of the epithelial cells to bind Corynebacterium renale and C. pilosum. C. renale adhered best to the epithelial cells of the vulva, and then to those of the ureter and renal pelvis. C. pilosum also adhered best to the epithelial cells of the vulva, followed by those of the vaginal vestibule. The results indicate that the most important target tissue for these bacteria may be the vulva, and the results correlate with the fact that C. renale frequently causes pyelonephritis and ureteritis, while C. pilosum causes the same diseases less frequently and behaves like normal flora of the vaginal vestibule.     

Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.

Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.     

抗鹅γ-干扰素单克隆抗体的制备及鉴定

IFN-γ也称免疫IFN,主要由CD4+ Th1细胞、CD8+T细胞和NK细胞产生,是体内具有抗病毒和免疫调节功能的重要细胞因子之一[1].IFN-γ不但具有抗病毒、抗肿瘤活性,也可作加强和改善机体 免疫应答的免疫佐剂,与病毒的保护性抗原基因共 表达可加强和改善机体免疫应答.在禽类IFN-γ的研究中,鸡、鸭等IFN-γ的相关性研究较深入,有 关鹅IFN-γ的研究报道较少[2-3].     

两株抗猪伪狂犬病毒的单克隆抗体制备与鉴定

采用猪伪狂犬病毒(PRV)ZJ01株纯化病毒免疫BALB/c小鼠,利用融合细胞技术和间接ELISA抗体筛选技术,制备并获得2株能稳定分泌抗PRV单克隆抗体的杂交瘤细胞株2B3和5C10,其中,2B3单抗为Ig G2a亚类,5C10为Ig G1亚类,轻链均为κ链。间接免疫荧光检测结果表明,2株单克隆抗体均能与PRV发生特异性反应。Western-blot结果表明,2B3单抗针对PRV g C蛋白,5C10单抗针对PRV g E蛋白。本研究为建立快速检测伪狂犬病毒感染的免疫学方法奠定了基础。     

Production and characterization of monoclonal antibodies against dog immunoglobulin isotypes

In this study, the effect of two oligodeoxynucleotide (ODN) sequences 5'GCT-AGA-CGT-TAG-CGT-3' (CpG-ODN) and 5'-GCT-AGA-GCT-TAG-GCT-3' (GpC-ODN) on the antigen-specific antibody and cellular immune response after intramuscular immunizations with OVA was analyzed in pigs. Pigs immunized with OVA supplemented with these ODNs showed a significantly enhanced primary antibody response in comparison with the control group which received OVA without ODN. This enhanced primary antibody response appeared ODN-sequence-independent as similar effects were seen in both ODN-groups. The OVA-specific antibody titers obtained after a single injection of antigen combined with either of both ODNs were as high as the titers in the control group after two injections. Furthermore, the ODN-supplemented animals showed significantly higher OVA-specific IgA antibodies in their saliva and nasal secretions at some time points after the first immunization. Proliferation assays showed that CpG- as well as GpC-ODN significantly enhanced the antigen-specific as well as the mitogen-induced proliferation in different lymphoid tissues. Furthermore, 48h after the third immunization the CpG-group showed a significantly decreased IL-6 mRNA expression in cells of the local draining lymph node but no significant difference in TGF-beta (Th3-like) and IL-10 (Th2-like). The ODN injected animals showed the tendency to have higher IFN-gamma (Th1-like) mRNA-expression in comparison with the control group. To our knowledge, these are the first in vivo studies in pigs, which demonstrate the appropriateness of CpG-ODN as immunostimulating adjuvants in vaccines for farm animals.     

Preparation and characterization of monoclonal antibodies against Mycoplasma bovis.

Monoclonal antibodies (mabs) against Mycoplasma (M.) bovis were prepared for use in diagnosis of bovine mastitis. From the original 32 hybridomas actively secreting mabs against M. bovis, 6 stable lines were cloned. Two of them, Mb 5D8 and Mb 4F6, recognized M. bovis antigens of estimated molecular weights of 33 and 26 kDa, respectively. They showed no cross-reaction to other bovine mycoplasmas, thus rendering them useful for specific detection of this pathogen. All mabs investigated cross-reacted with M. agalactiae which is known to be closely related to M. bovis, but does not occur in cattle. Two other mabs, Mb 5D4 and Mb 1F6, exhibited further cross-reactions to a number of bovine mycoplasma species. Finally, mabs Mb 5D5 and Mb 2G5 reacted with all mycoplasmas tested. The possibility that they recognized constituents of the broth culture medium is discussed.     

Preparation and characterization of monoclonal antibodies against an avian reovirus

Thirteen monoclonal antibodies against avian reovirus strain Uchida were derived. Of the 13 antibodies, three (MAb1, MAb2, and MAb3) had the ability to neutralize the infectivity of the virus. MAb1 neutralized strains Uchida, CS-108, and TS-142 equally. MAb2 neutralized the same three strains, but the activity of neutralization was 10 times higher against Uchida than against CS-108 and TS-142. MAb3 neutralized only strain Uchida. It seems that MAb1 and MAb2 have a rather broad neutralization activity and that MAb3 has a type-specific activity. These data indicate that there are both type-specific and more broadly specific neutralization epitopes in avian reovirus particles, as in mammalian reoviruses. In Western blot analysis, MAb1 bound to a sigma protein of strain Uchida, so it was suggested that this protein carries the epitope of the less specific neutralizing activity.     

Production and characterization of monoclonal antibodies against porcine interleukin-4

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system.