Isolation and characterization of factor I of the bovine complement system

OBJECTIVE: To isolate and characterize factor I of the bovine complement system. Sample Population-Serum obtained from the blood of beef cattle. PROCEDURES: Serum samples were fractionated to yield factor I by means of sequential precipitation, ion-exchange, and gel-filtration chromatography. The protein was identified throughout the procedure on the basis of its ability to degrade the alpha'-chain of bovine C3b in the presence of bovine factor H. Electrophoresis in polyacrylamide gels was used to assess the degradation of C3b and determine the molecular weights of factor I and its component polypeptide chains. RESULTS: Bovine factor I had an apparent molecular weight of 94 kd and consisted of 2 disulfide-bonded polypeptides that had apparent molecular weights of 51 and 42 kd (under reducing conditions). Factor H was required for the factor I cleavage of the alpha'-chain of bovine C3b into iC3b. A similar cofactor effect was provided by trypsinized bovine erythrocytes or erythrocyte ghosts. Bovine properdin was prepared and shown to be a single polypeptide chain of 58 kd in the reduced form. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine factor I can be purified from serum by a simple 4-step procedure. It is structurally and functionally comparable to factor I of other species, and its purification completes the isolation and characterization of all the soluble components of the bovine alternative complement pathway.     

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.     

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 eighth component of the bovine complement system

OBJECTIVE: To isolate and characterize the eighth component of the complement system (C8) in cattle. SAMPLE POPULATION: Fresh plasma obtained from beef cattle. PROCEDURES: Plasma samples were fractionated, using sequential precipitation and ion-exchange and gel-filtration chromatography, to yield C8. The protein was identified throughout the procedure on the basis of its hemolytic function. Electrophoresis in polyacrylamide gels was used to determine molecular weight and composition of polypeptide chains. Reconstitution of classical and alternative complement pathways was used to characterize the hemolytic function of bovine C8. RESULTS: The bovine C8 protein consisted of a disulfide-bonded alpha-gamma heterodimer that was noncovalently associated with a beta chain. Apparent molecular weight of the alpha, beta, and gamma chains under reducing conditions were 66, 61, and 23 kd, respectively. In the classical pathway of activation, bovine C8 and the ninth component of the complement system (C9) had species incompatibility with human C8 and C9 on sheep erythrocyte target cells. CONCLUSIONS: A simple 4-step fractionation procedure provided good yield of bovine C8 from plasma. The isolated protein was structurally comparable to C8 from other species. Purified bovine C8 may be useful in functional hemolytic assays to investigate the roles of complement-mediated lysis in the pathogenesis of inflammatory diseases and the killing of susceptible microorganisms.     

Bb fragment of bovine complement factor B: stimulation of the oxidative burst in bovine monocytes.

In a previous study, we reported that fragment Bb of bovine complement factor B activated bovine monocytes, as demonstrated by the uptake of 3H-deoxyglucose. In the present study, the effects of Bb on the production of superoxide anion and hydrogen peroxide by bovine monocytes was investigated. The production of superoxide was measured by the superoxide dismutase inhibitable reduction of cytochrome c. The change in absorbance was determined by a spectrophotometer at a wavelength of 550 nm. Hydrogen peroxide production was measured by the horse-radish peroxidase-dependent oxidation of phenol red. The resulting color change was measured by a spectrophotometer at a wavelength of 620 nm. Fragment Bb (20 micrograms/mL) induced the generation of 0.96 +/- 0.2 (mean +/- SEM) nanomoles of superoxide/2.5 x 10(5) monocytes at 5 min. The production of superoxide peaked at 15 min (1.24 +/- 0.3 nanomoles). The production of hydrogen peroxide was also rapid: 0.195 +/- 0.05 nanomoles/2.5 x 10(5) monocytes at 5 min with a peak at 15 min (0.250 +/- 0.04 nanomoles). These observations show that fragment Bb, which has serine protease activity, induces bovine monocytes to generate reactive oxygen intermediates such as superoxide and hydrogen peroxide.     

Fragment Bb of bovine complement factor B: stimulatory effect on the microbicidal activity of bovine monocytes.

We have previously shown that the Bb fragment of bovine complement factor B activates bovine monocytes and neutrophils. The activation was demonstrated by the enhanced uptake of 3H-deoxyglucose. To investigate the potential effect of fragment Bb on the microbicidal activity of bovine monocytes, a direct method was used. This method involves an initial ingestion period at 37 degrees C followed by repeated washing. The decrease in the total number of viable intracellular Staphylococcus aureus during the reincubation of the bacteria with bovine monocytes determines the intracellular killing. Maximal intracellular killing was seen when the monocytes containing the ingested S. aureus was incubated with fresh bovine serum (mean +/- SEM = 73.4 +/- 1.4%). On incubation of the monocytes, containing the ingested bacteria with heat-inactivated bovine serum, 32.5 +/- 0.7% of the intracellular bacteria were killed. When affinity-purified bovine factor Bb was added to the heat-inactivated serum, the intracellular killing capacity was almost restored (65.8 +/- 1.5%). When monocytes were incubated with medium alone, they killed 22.4% of the intracellular microorganisms. When fragment Bb (25 micrograms/mL) was added to the medium, the intracellular killing of S. aureus doubled (46 +/- 1.29%). We conclude that the Bb fragment of bovine complement factor B stimulates bovine monocytes in their microbicidal activity.     

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.     

牛源肠球菌的分离鉴定及毒力因子基因的检测

为探究牛源性肠球菌中6种毒力因子基因(ccf、asa1、ace、esp、cylA、gelE)的分布情况,采集了不同地区382份疑似隐形乳腺炎的奶样,常规分离纯化细菌,采用16SrRNA和16S~23SrRNA方法相结合的方法,共鉴定出了68株肠球菌并检测了其溶血特性和上述6种毒力基因的存在情况。结果显示,40株(58.82%)表现为α-溶血,10株(14.71%)为β-溶血,18株(26.47%)为γ-不溶血。68株(100%)携带asa1基因,49株(72.06%)携带gelE基因,43株(63.24%)携带ccf基因,12株(17.65%)携带ace基因,10株(14.71%)携带esp基因,6株(8.82%)携带cylA基因。54.41%的分离菌株同时携带gelE、ccf、asa1毒力基因,而青海民和分离株的esp与ace,gelE与ccf同时出现,有一定规律性;但溶血性基因cylA与14株溶血性肠球菌没有相关性。研究牛源肠球菌的溶血特性与毒力基因携带情况,对肠球菌性奶牛乳腺炎的防控具有指导意义,同时也为动物与人之间病原互相传播的研究提供试验依据。     

Alternative pathway of bovine complement: concentration of factor B, hemolytic activity and heritability

Concentrations of bovine factor B (Bbov) were determined by radial immunodiffusion in sera of 46 Holstein cows and heifers aged one to nine years. Mean values were 34.2 +/- 5.3 mg/100 ml. A hemolytic diffusion plate assay in agarose gel in presence of 10 mM EGTA and 5 mM Mg accurately measured concentrations of purified Bbov but gave higher mean values, i.e. 47.8 +/- 10.2 mg/100 ml, for concentrations of Bbov in whole sera. Hemolytic values obtained by the hemolytic diffusion plate assay, however, weakly correlated (r = .4539, p less than 0.01) with the serum concentration of Bbov measured by radial immunodiffusion. It was concluded that the hemolytic diffusion plate assay was not an accurate technique for the quantitative measurement of Bbov but a good assay for quantitation of the total hemolytic activity mediated via activation of the alternative complement pathway. It is suggested that the difference between the values obtained by the two tests for one particular serum is, to some degree, an expression of the ratio of amplification and restriction of the alternative pathway activity. No significant heritability (offspring and one parent) was detected for the hemolytic activity of serum. A heritability of 0.93 at a significance level of p less than 0.1 was determined for the serum concentration of Bbov.     

Host-dependent differential expression of factor H binding proteins, their affinity to factor H and complement evasion by Lyme and relapsing fever borreliae

Binding of complement factor H is crucial for the resistance of Borrelia to complement-mediated lysis. This study was aimed to assess the correlation between the expression of fH binding proteins (FHBPs) during the early phase of infection (48 h after the entry of Borrelia into the blood circulation) and complement resistance of the Borrelia genus. As expected, B. afzelii, B. burgdorferi sensu stricto and B. garinii (Serotype 4, PBi) showed resistance to complement mediated lysis when incubated with human and dog complement, which coincided with the significantly higher expression (P<0.05) of the FHBPs. Similarly, B. coriaceae showed resistance to cattle complement. In non-reservoir hosts borreliae failed to induce expression of FHBPs within 48 h of complement challenge, and did not survive. It is important to note that not only the expression of FHBP but also their binding to fH is required for borrelial resistance to the complement. fH binding may depend on the coiled-coil (CC) motifs observed in the FHBPs, especially at the C terminus. A loss of the C-terminal CC motif in BgCRASP-1 of SKT-1 strain was found in in-silico CC prediction, and may be coupled with SKT-1's inability to bind factor H and evade complement-mediated attack. In contrast, the C-terminal CC motif was observed (P - 1.0) in BgCRASP-1 of PBi that may contributed to its factor H binding and human complement resistance.     

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.     

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.     

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.     

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.