Paul A. Rebers

Procedure for determining heptose and hexose in lipopolysaccharides: Modification of the Cysteine-Sulfuric Acid Method

Abstract A modification is presented of the cysteine-H 2 SO 4 procedure for the quantitative assays of heptose and hexose in unhydrolyzed lipopolysaccharide of P. multocida . The modification overcomes the problems of interference and low sensitivity which we found in applying the heptose procedures of Dische and Osborn to unhydrolyzed lipopolysaccharide. We changed these procedures by ( a ) adding the cysteine-HCl to the reaction mixture before heating, ( b ) increasing the H 2 SO 4 concentration in the reaction mixture, ( c ) extending the heating period to 20 min, and ( d ) reading the absorbances at 1 hr. The resulting modification, when compared with the procedures of Dische and Osborn, gave the characteristic heptose absorbance peak with an increased differential absorbance of 1.7-fold for unhydrolyzed lipopolysaccharide of P. multocida and 2.5-fold for a commercial sample of E. coli lipopolysaccharide. d -Glycero- l -mannoheptose responded linearly over a 25–100 μg range with a 1.5-fold increase in sensitivity. Heptose in mixtures with hexoses gave a linear response with a 1.8-fold increase in detection. We also obtained a 2-fold increase in the determination of the primary cysteine-H 2 SO 4 product of glucose with our modification.

Fowl cholera: Cross-protective turkey antisera and IgG antibodies induced with Pasteurella multocida-infected tissue bacterins

Turkey antisera induced with formolized Pasteurella multocida-infected tissues (T antisera) passively cross-immunized 48 of 55 chickens against a challenge dose of P. multocida organisms, from which 0 of 15 controls survived. However, turkey antisera induced with formalin-killed, agar-cultured P. multocida cells (A antisera) passively cross-immunized only 4 of 30 chickens. Cross-immunity refers to protection against a different immunologic type of P. multocida. Quantitative precipitin reactions of the A and T antisera with antigens from agar-cultured cells showed that more antibody was present in the A than in the T antisera. However, antigens extracted from the infected tissues reacted with the T and not with the A antisera in the Ouchterlony procedure, demonstrating qualitative differences between the agar-cultured antigens and those extracted from the infected tissue. The gel precipitins isolated from the A and T antisera were characterized as 7S immunoglobulins, which behaved in immunoelectrophoresis as would be expected for a IgG immunoglobulin. The IgG fraction from the T antiserum passively cross-immunized chickens almost as well as the whole antiserum; hence, the IgG antibody is a major factor in cross-immunity.

The effect of formalin-killing of Pasteurella multocida on the antigenicity and extractability of its lipopolysaccharide

The extraction of lipopolysaccharides (LPS) from formalin-killed (FK) Pasteurella multocida strain X-73 and from cells not exposed to formalin (NF) were compared by the Westphal and phenol-chloroform-petroleum ether (PCP) extraction procedures. The LPS was determined by: (1) serologic analyses with antiserum specific for LPS; (2) analyses for toxicity; and (3) chemical analyses for components expected to be in LPS (such as hexoses, heptoses, amino sugars, 3-deoxyoctulosonic acid, and fatty acids). Strain X-73, the strain most virulent for chickens, was markedly affected by formalin killing. Unlike many strains, which readily yield LPS into the aqueous phase when extracted with phenol at 68 degrees by the Westphal procedure, strain X-73 did so only with FK and not with NF cells. With the NF cells, LPS was extracted by EDTA from the precipitate obtained during the Westphal procedure. With the PCP procedure, LPS was extracted readily from NF cells, but not from FK cells. The change in extractability of LPS as a result of formalin-killing was the same for both the encapsulated form of X-73 and a nonencapsulated variant derived from it. Although formalin-killing affected the extractability of LPS, no antigenic differences could be detected by immunodiffusion. However, the chick-embryo toxicity of LPS extracted from NF cells was greater than that of LPS from FK cells.