-Brucellosis control and eradication requires serological tests and vaccines. Effective classical vaccines (S19 in cattle and Rev 1 in small ruminants), however, induce antibodies to the O-polysaccharide of the lipopolysaccharide which may be difficult to distinguish from those resulting from infection and may thus complicate diagnosis. Rough attenuated mutants lack the O-polysaccharide and would solve this problem if eliciting protective immunity; the empirically obtained rough mutants 45/20 and RB51 have been used as vaccines. Strain 45/20 is reportedly unstable and it is not presently used. RB51 is increasingly used instead of S19 in some countries but it is rifampicin resistant and its effectiveness is controversial. Some controlled experiments have found good or absolute protection in adult cattle vaccinated orally (full dose) or subcutaneously (reduced dose) and in one field experiment, RB51 was reported to afford absolute protection to calves and to perform better than S19. Controlled experiments in calves, however, have shown reduced doses of RB51 to be ineffective, full doses only partially effective, and RB51 less effective than S19 against severe challenges. Moreover, other observations suggest that RB51 is ineffective when prevalence is high. RB51 is not useful in sheep and evidence in goats is preliminary and contradictory. Rough mutants obtained by molecular biology methods on the knowledge of the genetics and structure of Brucella lipopolysaccharide may offer alternatives. The B. abortus manB core (rfbK) mutant seems promising in cattle, and analyses in mice suggest that mutations affecting only the O-polysaccharide result in better vaccines than those affecting both core and O-polysaccharide. Possible uses of rough vaccines also include boosting immunity by revaccination but solid evidence on its effectiveness, safety and practicality is not available.

Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-D-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manB core to distinguish it from the wbk manB O-Ag . The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness.Brucellosis is a zoonotic disease that causes heavy economic losses and human suffering. Under most conditions, vaccination and serological identification and culling of infected animals are the only practical means to achieve its eradication, but the best vaccines available (Brucella abortus S19 for cattle and B. melitensis Rev1 for sheep and goats) may induce abortions when used in pregnant animals and are virulent for humans. Moreover, like field strains, they carry a cell surface smoothtype lipopolysaccharide (S-LPS) whose immunodominant section (the N-formylperosamine O-polysaccharide) induces an antibody response that may be difficult to distinguish from that resulting from a true infection (25,48). This complicates serodiagnosis because the tests currently used detect antibodies to the O-polysaccharide.To overcome these problems, several strategies are possible. The early observation that rough (R) B. abortus strains are attenuated and do not agglutinate with antibody elicited by S bacteria (63) soon led to the concept of Brucella R vaccines and, more than 50 years ago, the spontaneous R mutant B. abortus 45/20 was studied for this purpose. However, strain 45/20 was unstable, and its use was abandoned (1, 48). The same strategy was followed to devel...

Brucellosis is a disease caused by members of the genus Brucella that affects animals and humans. The species that infects cattle most often is Brucella abortus, but cattle infections by Brucella melitensis are not rare in areas where there is contact with infected sheep and goats (63, 64). Both B. abortus and B. melitensis are termed smooth (S) because they bear a S-type lipopolysaccharide (S-LPS). Many serological tests have been proposed for the diagnosis of brucellosis caused by S brucellae, and they can be broadly classified as those detecting antibodies to the S-LPS and those detecting antibodies to proteins (21,45). The former tests use either suspensions of S brucellae as antigens (3) or S-LPS extracts. The Rose Bengal test (RBT) and the complement fixation test (CFT) belong to the first group, and are recommended by the Office International des Épizooties for international trade (4). In addition, indirect enzyme-linked immunosorbent assays (ELISA) using S-LPS extracts or its O-chain have been extensively studied (47) and may replace the RBT and CFT. S-LPS tests are the most sensitive for detecting cattle brucellosis, but they may yield false positive results for cattle vaccinated with B. abortus

The peptide NK-2 is an effective antimicrobial agent with low hemolytic and cytotoxic activities and is thus a promising candidate for clinical applications. It comprises the ␣-helical, cationic core region of porcine NK-lysin a homolog of human granulysin and of amoebapores of pathogenic amoeba. Here we visualized the impact of NK-2 on Escherichia coli by electron microscopy and used NK-2 as a template for sequence variations to improve the peptide stability and activity and to gain insight into the structure/ function relationships. We synthesized 18 new peptides and tested their activities on seven Gram-negative and one Gram-positive bacterial strains, human erythrocytes, and HeLa cells. Although all peptides appeared unordered in buffer, those active against bacteria adopted an ␣-helical conformation in membrane-mimetic environments like trifluoroethanol and negatively charged phosphatidylglycerol (PG) liposomes that mimick the cytoplasmic membrane of bacteria. This conformation was not observed in the presence of liposomes consisting of zwitterionic phosphatidylcholine (PC) typical for the human cell plasma membrane. The interaction was paralleled by intercalation of these peptides into PG liposomes as determined by FRET spectroscopy. A comparative analysis between biological activity and the calculated peptide parameters revealed that the decisive factor for a broad spectrum activity is not the peptide overall hydrophobicity or amphipathicity, but the possession of a minimal positive net charge plus a highly amphipathic anchor point of only seven amino acid residues (two helical turns).

Increasing numbers of bacterial strains being resistant to conventional antibiotics emphasize the urgent need for new antimicrobial agents. One strategy is based on host defence peptides that can be found in every organism including humans. We have studied the antimicrobial peptide LF11, derived from the pepsin cleavage product of human lactoferrin, known for its antimicrobial and lipid A-binding activity, and peptide C12LF11, the N-lauryl-derivative of LF11, which has owing to the attached hydrocarbon chain an additional hydrophobic segment. The influence of this hydrocarbon chain on membrane selectivity was studied using model membranes composed of dipalmitoylphosphatidylglycerol (DPPG), mimicking bacterial plasma membranes, and of dipalmitoylphosphatidylcholine (DPPC), a model system for mammalian membranes. A variety of biophysical techniques was applied. Thereby, we found that LF11 did not affect DPPC bilayers and showed only moderate effects on DPPG membranes in accordance with its non-hemolytic and weak antimicrobial activity. In contrast, the introduction of the N-lauryl group caused significant changes in the phase behaviour and lipid chain packing in both model membrane systems. These findings correlate with the in vitro tests on methicillin resistant S. aureus, E. coli, P. aeruginosa and human red blood cells, showing increased biological activity of C12LF11 towards these test organisms. This provides evidence that both electrostatic and hydrophobic interactions are crucial for biological activity of antimicrobial peptides, whereas a certain balance between the two components has to be kept, in order not to loose the specificity for bacterial membranes.

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