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Complement is important for mediating opsonophagocytic killing of R equi , 23 , 30 and opsonization of R equi is important for intracellular killing of R equi . 29 , 31 – 35 Individual plasma donors with similar IgG concentrations can vary in their

Open access
in American Journal of Veterinary Research

SUMMARY

The activation of the complement system of rainbow trout by trout C-reactive protein (crp) was investigated. Complement fixation tests were performed by using rabbit hemolysin-sensitized sheep erythrocytes and rainbow trout complement. Purified crp increased the consumption of complement in the presence of Streptococcus pneumoniae C-polysaccharide (cps), indicating the activation of the complement system. In contrast to this, acute phase serum activated the complement in the absence of cps. Consumption of the complement by acute-phase serum was depressed when crp was removed from acute-phase serum by cps-sepharose 4B affinity chromatography. The acute-phase serum, as well as crp plus cps, suppressed in vitro growth of Vibrio anguillarum in the presence of complement, and enhanced the phagocytosis of the bacteria by glass-adherent peritoneal exudate cells. These results indicated that crp has a role in host defense during acute-phase response through the activation of the complement system, enhancement of phagocytosis, and suppression of bacterial growth.

Free access
in American Journal of Veterinary Research

Summary

The ability of polysulfated glycosaminoglycans (psgag) to inhibit the complement cascade was evaluated. The role of complement in inflammation and infection has been well documented. Inhibition of the complement cascade by psgag could explain why intra-articularly administered psgag diminish diarthrodial joint inflammation and potentiate septic arthritis in horses.

Hemolytic complement testing was performed to evaluate the effect of psgag on the equine classical and alternate pathways of complement, using rabbit erythrocytes as the target cells. Concentration of psgag between 0.2 mg/ml and 0.6 mg/ml significantly (P< 0.05) inhibited equine complement in dose-related fashion. Further increase in complement inhibition was not observed at psgag concentration >0.6 mg/ml. Difference was not apparent in the extent of inhibition of complement from each of the 4 horses tested. Polysulfated glycosaminoglycans appeared to inhibit the classical and alternate complement pathways equally, indicating possible effect on complement components common to both pathways. Heat inactivation of complement function completely inhibited (P<0.01) the hemolytic activity of the serum from all horses.

Free access
in American Journal of Veterinary Research

The complement cascade is a fundamental part of the host immune system that comprises > 20 serum proteins, many of which are proenzymes. 1 A functioning complement system is essential for immune function because it can eliminate invading

Full access
in American Journal of Veterinary Research

Abstract

Objective

To study binding of purified complement component C3b to bovine blood and mammary neutrophils (PMN) after various treatments and determine their ability to modulate receptor numbers.

Design

Cell isolation, activation, and flow cytometric studies.

Animals

Healthy lactating Holstein cattle.

Procedure

Complement component C3b (18,300 kd) was isolated from bovine serum by column chromatography, and flow cytometric assays using fluorescein isothiocyanate-labeled C3b were developed to evaluate binding to PMN complement receptor 1. Multiple substances were tested to determine their overall effect on C3b binding to PMN. Blood and milk PMN were isolated by differential centrifugation and exposed to optimal concentrations of recombinant human C5a, formyl-methyl leucyl phenylalanine, recombinant bovine interferon-γ, variable concentrations of phorbol myristate acetate (0.01 to 100 ng), calcium ionophore A23187, serum-opsonized zymosan, zymosan-activated serum (ZAS), zymosan-activated plasma (ZAP), and hydrocortisone acetate (25 and 70 ng). Additionally, mammary and blood PMN were preincubated in skim milk and whey.

Results

Variable concentrations of phorbol myristate acetate caused a dose-dependent increase in percentage of PMN binding C3b, and increased the amount of C3b bound per cell. Significant increases were observed after PMN treatment with calcium ionophore, serum opsonized zymosan, ZAS, and ZAP; conversely, incubation of PMN with hydrocortisone acetate resulted in reduced overall binding of C3b. Mammary PMN consistently bound more C3b, which was attributed to their activation during migration into the mammary gland. Binding of C3b was inhibited by skim milk. Activation of blood PMN with PMA, ZAS, and ZAP elicited larger responses than those observed for mammary PMN.

Conclusions

Modulation of complement receptors on bovine PMN is possible. Additionally, significant difference between the level of binding of C3b to blood and milk PMN, with milk PMN having higher binding, may be attributable to migration of PMN into the mammary gland, causing increased receptor expression.

Clinical Relevance

Contribution to a greater understanding of the role of complement in bovine immunologic systems, leading to testing for in vivo enhancement of bovine immune responses to invading pathogens.

Free access
in American Journal of Veterinary Research

infections were the CF and card agglutination tests. 12 The USDA CF method is a serum-dilution test based on hemolysis of erythrocytes as a result of fixation of complement. Titers are expressed in relation to the highest dilution of serum that has < 100

Full access
in American Journal of Veterinary Research

Summary

Conditions for purification of the ninth component of bovine complement (C9) were established. The conditions for binding and elution from diethylaminoethyl cellulose and hydroxylapatite were different than for human C9. Serum albumin, a frequent contaminant of bovine C9 preparations, was removed by chromatography on reactive-red agarose. The calculated molecular weight of bovine C9 was 66,000, and reduction with 2-mercaptoethanol affected its migration on polyacrylamide gel electrophoresis. Some preparations of bovine C9 migrated as 2 bands when partially reduced, but extensively reduced preparations had a single band.

Free access
in American Journal of Veterinary Research

SUMMARY

The possible role of the complement-mediated bactericidal system in protection of swine against contagious pleuropneumonia was investigated. Strains of Actinobacillus (Haemophilus) pleuropneumoniae representing serotypes 2, 3 and 5 were found to be fully resistant to the bactericidal action of porcine serum from precolostral, clinically normal adult, and chronically infected pigs. All strains were also resistant to hyperimmune rabbit serum, but 3 of 4 strains were sensitive to normal human serum. This bactericidal effect was lost when human serum was previously absorbed with the homologous bacteria, indicating that antibody was necessary for killing. Addition of human serum to porcine serum or to absorbed human serum did not restore the bactericidal system. Pretreatment of the bacteria with undiluted heat-treated human serum also failed to sensitize the bacteria to the absorbed serum, indicating that a heat-labile, absorbable factor may have been required for killing of A pleuropneumoniae. None of the strains was sensitized to porcine serum by sublethal treatment with polymyxin B, a treatment that is known to disrupt the integrity of the outer membrane and induce serum sensitivity in gram-negative bacteria. The ability of A pleuropneumoniae to resist complement killing in vitro may reflect a virulence mechanism in vivo that assists bacteria in avoiding the pulmonary defenses of swine and promotes bacterial invasion of the lungs.

Free access
in American Journal of Veterinary Research

Abstract

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 α-γ heterodimer that was noncovalently associated with a β chain. Apparent molecular weight of the α, β, and γ 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. (Am J Vet Res 1999;60:1474–1477)

Free access
in American Journal of Veterinary Research

Summary

Bovine immunoglobulin preparations from normal serum and from sera containing antibodies against Brucella abortus interfered with the brucellacidal action of bovine serum, whereas unfractionated normal serum and antisera were not inhibitory. The inhibitory property of immunoglobulin appeared to be attributable to some anticomplementary property because it also interfered with serum-mediated hemolysis of antibody-coated erythrocytes. The supernatant phase obtained after ultracentrifugation of bovine anti-B abortus immunoglobulin did not inhibit brucellacidal activity of normal bovine serum. Results of this study indicate that bovine anti-B abortus immunoglobulin preparations contain microaggregates of protein that can inhibit the ability of bovine serum to kill B abortus. The most likely mechanism is nonspecific activation of complement by microaggregated immunoglobulin, which consumes complement and makes it unavailable for bactericidal activity.

Free access
in American Journal of Veterinary Research