Evaluation of the opsonic capacity of core lipopolysaccharide antiserum of equine origin against smooth Escherichia coli 0111:B4, using macrophage chemiluminescence
Authors: Morris Debra Deem , Harmon Barry G. , Moore James N. 
Published: February 2025

A study was performed to determine whether equine antiserum to core lipopolysaccharide (lps) would enhance phagocytosis of smooth gram-negative (gn) organisms by equine macrophages. Five healthy adult horses (group A) were immunized with a bacterin prepared from the J-5 mutant of Escherichia coli 0111:B4 and Salmonella minnesota R595 to produce antibodies to core lps. Five horses (group B) served as nonimmunized controls and were given physiologic saline solution instead of the rough mutant bacterin. Serum antibody titers to core lps and to smooth E coli 0111:B4 were determined by indirect elisa. Four serum pools were prepared: pool 1 = sera from horses in group B prior to immunization; pool 2 = sera from horses in group A prior to immunization (preimmune serum); pool 3 = sera from horses in group B, 7 days after the last saline injection; pool 4 = sera from horses in group A, 7 days after the last immunization (core lps antiserum). The serum pools, either unheated or heated 30 minutes at 56 C, in 3 dilutions (1/50, 1/100, 1/500) were used to opsonize smooth E coli 0111:B4 in an assay of equine peritoneal macrophage chemiluminescence (cl). Peritoneal fluid was collected from clinically normal horses and the macrophages were purified by adherence to borosilicate glass scintillation vials. Each serum type and dilution was added to triplicate vials containing 107 colony-forming units of E coli 0111:B4. Luminol-dependent cl was measured with a liquid scintillation counter in the out-of-coincidence mode. Each serum dilution was tested in duplicate vials without bacteria to asses serum-induced nonspecific cl. Means of triplicate samples were calculated and numerical integrals were derived by use of trapezoidal approximation.

Core lps antibody titer in serum pool 4 (12,800) was substantially greater than that in pools 1, 2, and 3 (400). Titers to E coli 0111:B4 were not different among the 4 serum pools. When used at 1/500 dilution, unheated core lps antiserum (pool 4) was associated with greater total macrophage cl than was preimmune serum from the same horses (pool 2; P < 0.05). At all dilutions heat-inactivated core lps antiserum resulted in significantly greater total macrophage cl than did heated preimmune serum (P < 0.05). Heat inactivation of both sera (pools 2 and 4) at 1/50 dilution resulted in lower total macrophage cl and of pool 2 at 1/100 dilution. Dilution did not alter opsonic capacity of heated preimmune serum, whereas total cl associated with use of unheated preimmune serum was significantly affected by dilution of core lps antiserum did not affect macrophage cl, unless the former was heat-inactivated (P < 0.05). Total macrophage cl associated with use of serum pools 1 and 3 was similar to that produced, using pool 2. These data suggest that equine core lps antiserum has immuglobulin-mediated opsonic capacity for smooth gn organisms and may improve phagocytosis of the letter by equine macrophages.