Brucellosis is a zoonotic disease caused by members of the genus Brucella. These are non-spore-forming coccobacillary rods with cell wall characteristics of Gram-negative bacteria, which include peptidoglycans, outer membrane proteins, and lipopolysaccharide (LPS). The species Brucella abortus, Brucella melitensis, and Brucella suis cause the greatest animal and human health impacts. The LPS of field strains of these species possess O-polysaccharides (OPS), which protrude from the cell wall and alter the morphology of colonies giving rise to their description as "smooth" species and strains. The main feature of the disease in livestock is reproductive failure, which is most evident through abortion and male infertility (1). Otherwise, many animals with such an infection appear outwardly healthy. This is not so in humans, in whom undulant fever occurs in most cases (2).The principle method for monitoring brucellosis in the animal population, and a necessity for its eradication, is serology. The classical and contemporary methods for the serodiagnosis of brucellosis in animals have been well described (3-5), and despite differences, all the assays make use of diagnostic antigens that are rich in OPS.The main structural element within the Brucella OPS is a homopolymer of 4,6-dideoxy-4-formamido-D-mannopyranosyls (D-Rha4NFo), which are variably ␣(1¡2) and ␣(1¡3) linked (6-9). The proportion of each linkage type in different strains of Brucella appears to vary from 0% to 20% frequency of ␣(1¡3) linkage types, with the remainder being ␣(1¡2) types. Notably, only the B. suis biovar 2 type strain has been found to be devoid of ␣ (1¡3)

Brucellosis is diagnosed by detection of antibodies in the blood of animals and humans that are specific for two carbohydrate antigens, termed A and M, which are present concurrently in a single cell wall O-polysaccharide. Animal brucellosis vaccines contain these antigenic determinants, and consequently infected and vaccinated animals cannot be differentiated as both groups produce A and M specific antibodies. We hypothesized that chemical synthesis of a pure A vaccine would offer unique identification of infected animals by a synthetic M diagnostic antigen that would not react with antibodies generated by this vaccine. Two forms of the A antigen, a hexasaccharide and a heptasaccharide conjugated to tetanus toxoid via reducing and nonreducing terminal sugars, were synthesized and used as lead vaccine candidates. Mouse antibody profiles to these immunogens showed that to avoid reaction with diagnostic M antigen it was essential to maximize the induction of anti-A antibodies that bind internal oligosaccharide sequences and minimize production of antibodies directed toward the terminal nonreducing monosaccharide. This objective was achieved by conjugation of Brucella O-polysaccharide to tetanus toxoid via its periodate oxidized terminal nonreducing monosaccharide, thereby destroying terminal epitopes and focusing the antibody response on internal A epitopes. This establishes the method to resolve the decades-long challenge of how to create effective brucellosis vaccines without compromising diagnosis of infected animals.

No abstract

c Brucella abortus, a smooth strain of the genus Brucella, is the causative agent of bovine brucellosis. To support the ongoing development of diagnostic tests for bovine brucellosis, the use of Protein Saver cards (Whatman) for bovine blood serum and plasma sample collection has been evaluated. These cards offer significant logistical and safety alternatives to transporting and storing liquid samples and may aid in diagnostic programs and validation studies. To evaluate the utility of these cards, 204 bovine blood serum samples from Brucella-infected and noninfected animals were stored on and eluted from the Protein Saver cards. Anti-Brucella smooth lipopolysaccharide (sLPS) antibody titers for the serum eluates were compared to those of the unprocessed original serum samples by indirect enzyme-linked immunosorbent assay (ELISA). The results showed a highly significant correlation between titers from the serum eluates and the unprocessed sera. Therefore, under these circumstances, serum eluates and unprocessed serum samples may be used interchangeably. Blood plasma from 113 mitogen-stimulated whole-blood samples was added to and eluted from the Protein Saver cards. The gamma interferon (IFN-␥) titers in the plasma eluates were compared to those of the unprocessed plasma samples obtained by IFN-␥ ELISA. The results showed a significant correlation between the plasma eluates and the unprocessed plasma samples. To derive a signal in the plasma eluate, it was necessary to develop a novel and highly sensitive ELISA for the detection of IFN-␥. The serum samples stored on cards at room temperature over a 10-day period showed little variation in antibody titers. However, the plasma eluates showed a progressive loss of IFN-␥ recovery over 10 days when stored at room temperature.

The World Organisation for Animal Health (OIE) requested an International Standard anti-Brucella melitensis Serum (ISaBmS) to standardise diagnostic tests and reagents for sheep and goats. The agreed criteria were the highest dilution (in negative serum) of the standard which must give a positive result and the lowest dilution (in negative serum) which must simultaneously give a negative result. The two dilutions for each assay were, respectively: indirect enzyme-linked immunosorbent assay (iELISA) 1/64 and 1/750, competitive ELISA (cELISA) 1/8 and 1/300, fluorescent polarisation assay (FPA) 1/16 and 1/200, Rose Bengal test (RBT) 1/16 and 1/200. The OIE International Standard Serum (OIEISS) will remain the primary standard for the RBT; the ISaBmS is an additional standard. It was impossible to set criteria for the complement fixation test, therefore the OIEISS will remain the primary standard. The ISaBmS can be used to standardise iELISA, cELISA and FPA to diagnose sheep and goat brucellosis. This standard should facilitate harmonisation of tests used for brucellosis surveillance and international trade in these species.