A simple hemolytic assay for bovine complement component C3

A simple, one-step, alternative pathway (AP) hemolytic assay for bovine C3 has been developed. Methylamine was used to prepare a bovine serum reagent, R3, functionally depleted of C3. The addition of purified bovine C3 to the R3 reconstituted, in a dose-dependent manner, the hemolytic activity for unsensitized heterologous erythrocytes. The assay was used to determine relative levels of C3 in different bovine serum samples. Human C3 and bovine C3 were interchangeable in the assay. Reconstitution of bovine and human R3 reagents with homologous or heterologous C3, in the presence of different species of erythrocytes, provided evidence that cell surface regulation of the homologous hemolytic AP may not be limited to the assembly and activity of the C3 convertase. The AP assay was more sensitive and less complex to perform than a standard classical pathway assay for bovine C3.     

Degradation of bovine C3 by serine proteases from parasites Hypoderma lineatum (Diptera, Oestridae)

Purified enzymes of Hypoderma lineatum (Insecta, Oestridae), were assayed for their proteolytic activity on bovine C3 in normal cattle sera. The products of cleavage by these serine proteases (hypodermins A, B, and C), were analysed by electrophoresis in SDS polyacrylamide gels followed by immunoblotting. The enzymatic attack was initially directed at the alpha polypeptide chain by hypodermin A at a concentration of 1 μg/ml of serum and by hypodermin B at 5 μg/ml. The generated peptides differed in their molecular size from those produced during natural degradation of C3 in a control serum by physiologically relevant enzymes. Hypodermin A, at a concentration of 1 μg/ml, also caused a cleavage of the β chain. At 5 μg/ml, hypodermin A induced total degradation of the C3 molecule. Hypodermin B (5 μg/ml) starts splitting C3 near cleavage sites of factor I. Bovine C3 appears to be highly sensitive to hypodermins A and B in normal sera. Apparent molecular sizes and alignment of the bovine C3 cleavage products are presented schematically. Hypodermin C, a collagenolytic enzyme, had no effect on C3 in normal sera. The biological consequences for the immunopathological reactions associated with hypodermosis are discussed.     

Hemolytic complement titers and complement C3 levels in endotoxin-induced mastitis

Escherichia coli lipopolysaccharide B was instilled through the lactiferous duct of cows to induce acute mastitis. Hemolytic complement (C) activity and C3 concentrations were determined in blood serum and in renninprecipitated whey before, and at certain times after, mastitis was induced. Hemolytic complement activity was detected in the whey only during the first 36 hours after endotoxin was instilled, whereas activity was not seen before and 48 or more hours after the endotoxin was given. The maximum titer as measured with the guinea pig RBC/bovine natural antibody system was 1:64. The C3 concentrations in normal whey (before installation of endotoxin), measured by radial immunodiffusion, were between 1% and 4% of the base-line blood serum values (pool from healthy cows). The whey concentration of C3 increased (to 5% to 18%) during the first 8 hours of mastitis. However, at 72 hours, the whey values were back to preinstillation concentrations in all quarters.     

Activation of complement by bovine respiratory syncytial virus-infected cells

Because complement activation is probably involved in the pathogenesis of as well as in recovery from the disease induced by bovine respiratory syncytial virus (BRSV), we studied the activation of complement by BRSV-infected cells in vitro in a homologous system. Binding of C3 on the surface of infected cells was measured in a biotin-streptavidin amplified ELISA, and complement-mediated lysis was measured in a 51Cr release assay. Without antibody, infected cells activated and bound more C3 than uninfected cells. C3 activation that occurred in the absence of antibody was largely mediated by the classical pathway and induced lysis inefficiently. BRSV-specific antibody enhanced complement activation as measured by both C3 ELISA and cytotoxicity assay. In the presence of antibody, C3 activation was largely dependent on the alternative pathway and efficiently induced lysis. Both IgG1 and IgM antibodies enhanced C3 activation, but IgG2 and IgA did not enhance C3 activation in our experiments. Preincubating cells with IgA or IgG2 did not inhibit C3 activation enhanced by IgG1 or IgM. Murine monoclonal IgG1 antibodies against epitopes on the Fusion protein of the virus also enhanced C3 binding, but differed in their capacity to induce complement-mediated lysis.     

Differential complement activation by bovine IgG2 allotypes.

Immunoglobulin allotypes and complement (C) are known to be related to susceptibility to infection. Because bovine IgG2 is important in resistance to pyogenic infections and because its two allotypes, IgG2a and IgG2b, differ in sequence in the CH1, hinge, CH2, and CH3 regions, we tested the ability of these allotypes to initiate the bovine C cascade. Bovine IgG2a and IgG2b were standardized according to specific anti guinea pig red blood cell (GPRBC) ELISA activity using anti IgG2 reagents shown essentially unbiased for allotype. Complement activating activity of the allotypes was quantitated in a GPRBC lysis assay. With this system, IgG2b consistently had more than twice the activity in bovine C mediated lysis as compared with IgG2a. The fact that both EDTA and EGTA/Mg almost completely inhibited C mediated lysis of GPRBCs indicated that lysis was due to the classical pathway. Since antibody usually activates C by the classical pathway, this supports the supposition that activation was by the IgG2-GPRBC complexes. Flexibility analyses showed that IgG2b had a more rigid hinge than IgG2a, perhaps partially explaining the greater efficiency of IgG2b in C activation. Other mechanisms may include differences in glycosylation and in the amino acid at position 332. The difference in ability to activate C may mean that animals of the IgG2a allotype could be more susceptible to infection with extracellular pyogenic pathogens which are killed by C or by phagocytes after opsonization with IgG2 and C.     

抗鸡补体C3抗体的制备及其应用

选取鸡源补体C3a链的21个氨基酸残基序列，人工合成模拟C3分子的21肽^[741-761]，与牛血清白蛋白载体偶联结合后，制成人工合成抗原复合物。将此人工合成抗原加弗氏佐剂制成油乳剂疫苗，免疫注射家兔制备成抗21肽抗体。从抗血清中提纯IgG，用以检测不同动物血清中C3含量。结果表明，人工合成的c3分子的21肽复合抗原具有很强的免疫原性，制成的抗血清的IgG效价达2。，且能与不同动物血液的c3分子发生特异性结合；用该IgG对鸡、猪、山羊、牛和豚鼠体内的C3进行了检测，其含量分别为（0．34±0．005）、（1．47±0．008）、（1．70±0．005）、（1．78±0．007）、（4．18±0．008）g／L，发现鸡体内的C3含量明显低于其他几种动物，差异极显著（P〈0．01），这可能是鸡体对细菌性疫苗免疫效果不佳的主要原因。     

Cloning of a gene fragment encoding bovine complement component C3d with expression and characterization of derived fusion proteins

The gene fragment coding for bovine C3d gene (boC3d) was cloned and expressed as a component of fusion proteins destined for use in vaccine studies in cattle, and for in vitro experiments. This fragment of complement protein C3 (C3d) has been shown to enhance B cell responses when complexed with antigen. Three potential vaccine constructs were engineered to contain one, two or three boC3d units linked to a fragment of the leukotoxin of Mannheimia haemolytica A1, an economically important pathogen of cattle that causes a fibrinous pneumonia in calves. A recombinant biotinylated boC3d protein (for use in in vitro studies) was generated by endogenous biotinylation in Escherichia coli by means of the BirA holoenzyme synthetase. All recombinant proteins incorporated polyhistidine tags and were purified by nickel-agarose chromatography, then analyzed by SDS-PAGE and Western immunoblot. The identity of boC3d was confirmed by mass spectrometry, since monoclonal antibodies to boC3d were not available. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice and rabbits, using antigens unrelated to diseases occurring naturally in these species. The boC3d fusion proteins expressed in this study will provide the basis for immunization trials in cattle and studies of receptor binding and cell activation of bovine lymphocytes.     

Interaction of bovine immunoglobulins with complement

Whereas complement (C) in rabbit serum (CR) was bound by bovine antibodies in seven different IgG1 preparations, only two IgG1 preparations could bind the C in guinea pig serum (CGP). Addition of the Clq component of CR to CGP was alone sufficient to render the C-cascade in CGP activable in the presence of bovine erythrocytes sensitized with specific antisera, i.e. reagents. Normal bovine serum was also capable of restoring the haemolytic activity of CGP. However, the bovine serum was much more temperature sensitive than was CR and, as was observed in the sera from MZ twins, it showed considerable variation both in titre values and in prozones when added to CGP.     

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.     

Mycoplasmacidal action of bovine complement mediated by bovine IgG1, IgG2 or IgM

The effect of several inhibitors of complement were examined in haemolytic, bactericidal and myoplasmacidal systems with bovine serum as the complement source. It appears that although EGTA-Mg allows the alternative complement pathway to function in a haemolytic system it has an inhibitory effect on this pathway in bactericidal and mycoplasmacidal systems. Both ?-aminocaproic acid and salcyladoxime were found to be useful for distinguishing the complement pathways in bovine serum and the results of experiments with these substances indicated that bovine IgG1 and IgG2 activated bovine complement, with a mycoplasma as the target cell, by the classical pathway. Mycoplasma bovis, which unlike Acholeplasma laidlawii, does not activate the alternate pathway in gnotobiotic-calf serum, was killed by serum from cattle that had not been infected previously with this mycoplasma. In this case killing was apparently mediated by cross-reacting IgM and complement via the classical pathway.     

A simple isolation procedure for functionally pure components of the bovine alternative complement pathway (ACP) C3 convertase and bovine conglutinin (K)

A simple multicomponent isolation procedure for bovine C3, factor B, factor D and conglutinin (K) from a single serum sample is described. The components of the alternative pathway C3 convertase were isolated in milligram quantities from 800 ml bovine serum and were found to be functionally pure with respect to each other and to factors H and I.     

Serum haemolytic complement activity and C3 levels in bovine trypanosomosis under natural conditions of challenge--early indications of individual susceptibility to disease

Twenty-five Baoule (Bos taurus) and 12 Zebu (Bos indicus) cattle, which were part of an experiment aimed at characterizing cattle for resistance to trypanosomosis under natural challenge in Burkina Faso, were monitored for complement levels. Total haemolytic activity of the alternative complement pathway and C3 in sera taken weekly were estimated. The results were analysed in relation to the course of the disease, parasitological data, packed red cell volume (PCV) and body weight. All the animals became infected with Trypanosoma vivax and/or T. congolense. The Zebu had to be treated with Berenil (Diminazene aceturate, Hoechst, W. Germany) after a mean period of 5 weeks of infection, whereas 7 of the 25 Baoule remained in good condition throughout the experiment. The remaining 18 Baoule required treatment after a variable period of infection. There was a decrease in haemolytic complement activity (HC') as well as in C3 levels, which coincided with the first detection of parasites in the blood. The titres in the Zebu fell to 10-20% of pre-infection level within 2-3 weeks and they showed no tendency towards regaining normal levels. The drop in complement in the Baoule was less pronounced and was in most cases followed by an increase approaching normal values. In these animals, the complement level in early infection was found to depend on the intensity of parasite load and on the control potential of each individual. There was a significant correlation between minimum complement activity (min. HC'), minimum C3 (min C3) and minimum PCV (min.PCV) in early infection. These three parameters correlated with individual resistance and might, therefore, be useful criteria for the identification of the most resistant individuals within a trypanotolerant breed.     

Neutrophil extracellular trap formation by bovine neutrophils is not inhibited by milk

Neutrophils are the first line of defense in a mammary gland infection. However, the process of neutrophil transmigration across a membrane and ingestion of fat and/or casein when incubated in milk have been shown to inhibit bacterial phagocytosis and oxidative burst functions. Recently, a killing mechanism has been described whereby stimulated neutrophils release nuclear and granule material in fibrous webs that physically trap and kill bacteria. We demonstrate that these neutrophil extracellular traps are also produced by bovine blood neutrophils stimulated with PMA/ionomycin. Importantly, neutrophil extracellular traps can be formed when neutrophils have been incubated for up to 6h in milk prior to stimulation. This contrasts milk's rapid inhibition of bacterial phagocytosis and oxidative burst functions in the neutrophil. Furthermore, stimulation of neutrophils with bacteria common to mammary gland infections leads to neutrophil extracellular traps being formed in milk. Some bacteria tested stimulated enhanced formation of neutrophil extracellular traps in milk compared to culture media. Therefore, being unaffected by incubation in milk may indicate an important role for neutrophil extracellular traps in defense against mastitis.     

复合菌群LF对DOP降解特性研究

从河底污泥样品中分离培养得到一株能以DOP为唯一碳源和能源的复合菌群,命名为LF,菌群LF能够高效降解DOP。本研究主要从pH、温度、含盐量、加菌量和底物初始浓度等五个方面确定富集物LF对DOP的最适降解条件,并考察菌群LF对DOP的降解性能。实验结果显示:LF降解DOP的最适pH为6.0,最适温度为30℃;在此最适pH、最适温度条件下,含盐量为1%时,菌群LF对DOP具有较高的降解活性;48 h内,菌体数量为6%时,菌LF降解率最高,为98%左右;72 h内富集物LF对不同初始底物浓度DOP一直保持有较高的降解率,初始浓度为1000 mg/L的底物DOP降解率最高。     

Activation of bovine monocytes and neutrophils by the Bb fragment of complement factor B: demonstration by the uptake of 3H-deoxyglucose.

The Bb fragment is the enzymatically active split product of bovine complement factor B. The Bb fragment was obtained after zymosan treatment of fresh bovine serum and fractionation of the treated serum, first over diethylaminoethyl-Sephacel and then over an affinity column made up of monoclonal antibody to bovine Bb, coupled to cyanogen-bromide-activated Sepharose. Purified Bb has a molecular weight of 64,000, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The ability of purified Bb to activate phagocytes was assessed. The activation assay was based on the principle that the primary source of energy for the phagocytes is obtained from glucose. 3H-deoxyglucose, a nonmetabolizable analogue of glucose, was used to obtain the quantitative measurement of the activation process. The activation by Bb was shown by the uptake of the labelled deoxyglucose in the phagocytic cells and was comparable to the activation caused by phorbol myristate acetate and N-formyl-L-methionyl-L-leucyl-L-phenylalanine, run in parallel. These data showed that fragment Bb activates bovine monocytes and neutrophils and also suggested that, when generated after complement activation, Bb may stimulate monocytes and neutrophils for enhanced phagocytosis.     

Failure of bovine complement levels measured by radial hemolysis to correlate with tube titration

Hemolytic complement levels in 30 duplicate samples of normal bovine sera were determined by a tube titration method (CH50) and by a radial hemolysis in gel assay. Significant correlation was not observed between the values obtained by the 2 tests. This lack of correlation could be a result of considerable error observed in values obtained for duplicate samples in the CH50 method or to the relative insensitivity of the hemolysis in gel test. The hemolytic reaction in gel was found to depend on Mg2+, thus raising questions (i) about its validity in determining total hemolytic complement or (ii) about bovine C1q depending on Mg2+ rather than Ca2+ for binding to immune complexes.     

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.     

Isolation and characterization of the third component of bovine complement

C3 was obtained from bovine serum by polyethylene glycol precipitation and chromatography on DEAE-Sephadex A-50, CM-Sephadex A-50 and Sephacryl S-200. The protein has a molecular weight of 183,000 (alpha-chain 114,000 and beta-chain 69,000). A CVF-induced bovine C3 convertase (Sepharose-CVF.Bb) cleaved C3 into C3a (11,000) and C3b (172,000) as shown by SDS-polyacrylamide gel electrophoresis. Isoelectricfocusing of C3 demonstrated at least three electrophoretic variants with pI 6.55–6.85. The isolated protein promoted the formation and action of a C3 convertase in the presence of purified bovine factors B and D. A monospecific antiserum prepared in rabbits failed to cross react with human C3 or CVF. C3c was identified as a contaminant during the isolation of C3.