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The effect of meningococcal cell-associated sialic acid on activation of the human alternative complement pathway was examined by using a quantitative fluorescence immunoassay to assess alternative pathway-mediated C3 binding to a group B strain of Neisseria meningitidis from which graded amounts of sialic acid had been removed with neuraminidase. Using human serum absorbed with strain B16B6 (B:2a:L2,3) and chelated with 10 mM MgCl2 and 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we found an increase in the amount of C3 bound by enzymatically desialylated B16B6 organisms over the amount bound by fully sialylated organisms. This increase was proportional to the amount of sialic acid cleaved from the bacteria. Enhanced C3 binding was accompanied by an increase in factor B deposition. A sialic acid-deficient mutant of strain B16B6, designated 2T4-1, bound C3 via the alternative pathway at a level equivalent to that bound by wild-type meningococci from which 88% of the sialic acid had been removed. Strain B16B6 was resistant to the alternative pathway-mediated bactericidal activity of both absorbed and hypogammaglobulinemic human sera, whereas noncapsular variant 2T4-1 was sensitive to these sera. The addition of purified immune immunoglobulin M (IgM) and IgG significantly increased the alternative pathway-mediated killing of strain B16B6 organisms. IgM mediated increased bactericidal activity without an increase in C3 or factor B deposition. In contrast, the IgG-mediated killing was associated with increased binding of C3 and factor B to the organisms. Absorption studies showed that the IgM bound to the sialic acid capsule, whereas the IgG bound to noncapsular surface antigens. We conclude from these results that the group B meningococcal sialic acid capsule inhibits activation of the alternative pathway in the nonimmune host and that both IgM and IgG, although specific for different surface antigens, are capable of augmenting the alternative pathway-mediated killing of group B meningococci.

Deoxyribonucleic acid (DNA) base composition, intergenic transformation efficiency, and DNA hybridization were used to determine the relatedness of a variety of established or proposed species of Neisseria and Branhamella. These studies indicated that these bacteria form three genetic groupings. Group I, comprised of N . meningitidis, N . gonorrhoeae, N . subflava, N . flava, N . perflava, N . sicca, N . mucosa, N . cinerea, N . flavescens, N . lactamica, N . elongata, N . canis, and N . denitrificans, was characterized by DNA base compositions ranging between 49.3 and 55.6 mol% guanine plus cytosine. Group 11, comprised of N . cuniculi, N . caviae, and N . ovis, was characterized by DNA base compositions ranging between 45.3 and 47.3 mol% guanine plus cytosine. Group 111, comprised of one species, B . catarrhalis, was characterized by DNA base compositions between 41 and 42 mol% guanine plus cytosine. Transformation and DNA hybridization results revealed that members of each group, with few exceptions, exhibited high DNA homology with other members of the same group but most often distinctly lower levels of homology with members of a different group. These data suggest that N . ovis, N . caviae, and N . cuniculi may be significantly different from other neisseriae and from branhamellae to warrant their separation in a distinct genus. The results of nucleic acid hybridizations performed by Kingsbury (17) revealed that the genus Neisseria was a heterogeneous group comprised of at least three distinct subgroups and questioned the proper classification of N . catarrhalis and N . caviae as members of the genus Neisseria. On the basis of nucleic acid hybridization studies, Bdvre reported that N . ovis, N . caviae, and B. catarrhalis showed distinct degrees of relatedness to several Moraxella species (3, 4). This supported the inclusion of Moraxella in the family Neisseriaceae.The purpose of this study was to use DNA base composition determinations, transformation, and DNA hybridization studies to clarify the taxonomic positions of recognized or proposed species of Neisseria and Branhamella. Special attention was focused on evaluating the potential of transformation as a toxonomic tool for determining the relatedness of members of these genera. MATERIALS AND METHODSThe bacterial strains examined are listed in Table 1. Stock cultures were preserved by freeze-drying. Working cultures were maintained at -70°C in Trypticase soy broth (BBL Microbiology Systems) supplemented with 6% lactose. Cultures were routinely passaged on GC agar consisting of GC medium base (Difco Laboratories) and 1 % (vol/vol) chemically defined supplements (36) and were incubated at 36°C for 18 h (5% C02, humidity).Preparation of DNA for base composition determinations. A modification of the procedure originally described by Marmur (25) was followed for the extrac- 57

A Leptospira species is suspected of being the etiological agent in a recent epizootic among California sea lions. The disease was confined to subadult males of the species Zalophus c. californianus .

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