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Brucellae are facultative intracellular gram-negative bacteria that cause human disease and significant worldwide economic loss due to infection of livestock. Live attenuated Brucella abortus S19 and Brucella melitensis Rev.1 have served as efficacious vaccine strains for cattle and sheep, respectively (19). Current serologic tests are the major tools for brucellosis diagnosis and mainly detect antilipopolysaccharide antibodies. This dominant antigen is common to virulent and vaccine strains. Therefore, the distinction between infection and vaccination is difficult to make.

Over the past few years studies have been conducted on antiprotein antibody response elicited during brucellosis to identify potential diagnostic antigens (8, 9, 16-18, 21, 22). It appeared that the antibody response against most of the proteins identified was heterogeneous among infected animals and that only a combination of selected Brucella proteins could lead to a sensitive diagnostic test.

Another approach is based on the measure of the specific cellular immune response in infected animals. The delayed-type hypersensitivity (DTH) assay is extremely specific and is complementary to the serologic diagnosis of bovine brucellosis (2, 13). More recently, the gamma interferon (IFN-) assay was found to be a powerful diagnostic tool (23). The production of an allergen of defined composition could contribute to the improvement of the DTH test or the IFN- assay.

The P39 protein is one of the major components of the allergen manufactured by Rhône-Mérieux, Lyon, France (brucellergene). A brucellergene fraction containing the P39 induced a positive DTH reaction in infected guinea pigs and stimulated the production of IFN- by blood cells of infected cattle (12). In cows, DTH and lymphoblastogenesis tests with purified P39 seemed to be specific and sensitive (11). The gene encoding P39 has consequently been cloned and sequenced (11). Purified recombinant P39 also seemed to be a promising antigen for the serologic diagnosis of animal brucellosis (17). Thus, P39 appeared to be useful for the detection of both humoral and cellular immune responses of infected animals.

In the present report, we describe the deletion of the P39 gene from Brucella vaccine strains S19 and Rev.1 and the effect of this deletion on residual virulence and protection in a mouse model. Animals vaccinated with such an engineered vaccine strain would not develop an immune response to P39, and P39 could be further used as an antigen for the differentiation of vaccinated and infected animals.

Construction of P39 gene deletion mutants of B. abortus S19 and B. melitensis Rev.1. Construction of the deletion plasmid used for the P39 gene replacement in Brucella was done as follows (Fig. 1A). A 1.65-kb EcoRI-XbaI fragment encoding P39 was excised from pTZ1.2. (11) and cloned into the vector pBluescript SK() (Stratagene, La Jolla, Calif.) to create p396. The P39 gene open reading frame was deleted from a 1,008-nucleotide fragment by digestion of p396 at the BsmI and BglII unique sites. DNA ends were made blunt, ligated to BamHI linkers, digested with BamHI, and then ligated to the 1.3-kb BamHI kanamycin resistance cassette (kan) from vector pUC4K (Pharmacia P-L Biochemicals, Uppsala, Sweden). This generated the plasmid pD391. A 0.76-kb EcoRI fragment containing oriRK2 was excised from pTJS82 (kindly provided by G. Cornelis, Microbial Pathogenesis Unit, Institute of Cellular Pathology, Brussels, Belgium) and ligated into the EcoRI site of pD391, generating the deletion plasmid pD392. This plasmid was conjugated from Escherichia coli S17-1 into a variant of B. abortus S19 which is resistant to nalidixic acid (Nal^r) and B. melitensis Rev.1 Nal^r. Since pD392 is unable to replicate in Brucella, the vector-borne kan gene should be rescued by homologous recombination. A double crossover due to homologous recombination events in each of the P39 gene flanking arms resulted in replacement of the P39 gene coding sequence by the kan marker and loss of the vector-encoded bla gene. Brucella transconjugants were selected in the presence of nalidixic acid and kanamycin and further screened by replica plating for ampicillin-sensitive colonies. One Nal^r, kanamycin-resistant, and ampicillin-sensitive colony of each vaccine strain was chosen for further study, and the strains were named S19P39 and Rev.1P39.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Construction of P39 deletion mutants by gene replacement. (A) Schematic restriction map of plasmid p396 and pD391 inserts. Black hatched box, P39 gene open reading frame; white hatched box, P39 gene probe. X, XbaI, B, BamHI; Bs, BsmI; H, HindIII; Bg, BglII; E, EcoRI. Arrows represent the direction of translation. (B) Southern hybridization of HindIII-digested parent and mutant genomic DNA with P39 gene or kan probes. Lanes 1, Rev.1; lanes 2, Rev.1P39; lanes 3, S19; lanes 4, S19P39; lanes MW, biotinylated lambda/HindIII and X174/HaeIII digests.

Residual virulence of the P39 gene mutants in a mouse model. In order to determine the residual virulence of the P39 gene mutants compared to that of the parent strains, 6-week-old CD-1 female mice (eight per group) were injected subcutaneously with 0.2 ml of phosphate-buffered saline (PBS) containing 1.2 × 10⁸ CFU of either B. abortus S19 or S19P39 or B. melitensis Rev.1 or Rev.1P39 (7). Mice were killed at 1, 3, 6, 9, 12, and 15 weeks after the challenge. Their spleens were homogenized in PBS, serially diluted, and plated on tryptic soy agar-yeast extract (TSA-YE). The numbers of CFU per organ were expressed as the log CFU to normalize the distribution of individual counts required for variance analysis (7). Means and standard deviations of transformed values per group were then computed. The 50% recovery times (RT₅₀) and confidence limits (P = 0.95) were calculated at the end of the experiment from the accumulated numbers of Brucella-free spleens by the plotted probit method of Bonet-Maury et al. (3). The P39 gene replacement appeared to be stable because bacterial colonies recovered from mouse spleen at different times postinjection were found to retain kanamycin resistance. Brucella counts in spleens from mice injected with strain Rev.1P39 decreased as regularly as counts of strain Rev.1 from week 3 to week 12 (Fig. 2A). Although the numbers of Brucella-infected spleens were similar at weeks 1, 3, 6, and 9 for both strains, strain Rev.1P39 counts were higher than those of strain Rev.1. Three mice were still infected with Rev.1 at week 15, whereas all mice injected with strain Rev.1P39 were Brucella free. RT₅₀ calculated at the end of the experiment were 10.3 weeks for Rev.1 and 8.2 weeks for Rev.1P39, with confidence limits of ±2.3 and ±2.7, respectively. The difference in RT₅₀ between the two strains was not statistically significant.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Residual virulence of B. melitensis Rev.1 strains (A) and B. abortus S19 strains (B). Spleen infection of CD-1 mice after subcutaneous injection of B. melitensis Rev.1 () and Rev.1P39 () and B. abortus S19 () and S19P39 (). Shown are individual results (symbols), mean time courses (lines), and RT50 (horizontal bars, with confidence limits, for mice receiving parent [black bars] and mutant [white bars] strains). Numbers in parentheses are numbers of negative mice out of eight receiving parent strains (bold numbers) and mutant strains (lightface numbers).

Protection conferred by the B. abortus and B. melitensis vaccine strains with P39 gene deletions in CD-1 mice against the relevant virulent challenge. P39 deletion mutants as well as the parent strains were tested in the CD-1 mouse model (5, 20) for their ability to protect against a virulent challenge. Mutant vaccine strains (10⁵ CFU/0.2 ml), parent vaccine strains (10⁵ CFU/0.2 ml) as positive controls, and PBS (0.2 ml) as a negative control were injected subcutaneously into 12 mice per group. Thirty days later, the virulent challenge strain (2 × 10⁵ CFU of B. abortus 544 or 1 × 10⁴ CFU of B. melitensis H38) was administered by the intraperitoneal route. Six mice from each group were randomly killed by cervical dislocation to isolate the spleens, 2 or 8 weeks postchallenge. Each spleen was weighed, homogenized, diluted, and spread on TSA-YE alone or TSA-YE plus 0.1% erythritol for differentiation of both B. abortus S19 strains from 544 or on TSA-YE containing 2.5 µg of streptomycin per ml for differentiation of both B. melitensis Rev.1 strains from H38 (S19 and 544 were also differentiated on the basis of CO₂ requirement) (1). Colonies of Brucella were enumerated. The number of CFU per spleen was then transformed to y = log(x/log x). This transformation normalizes the distribution of individual counts as required for variance analysis (5, 6).

View larger version (50K): [in this window] [in a new window]   FIG. 3.   Immune protection conferred in CD-1 mice by B. abortus S19 strains against a B. abortus 544 challenge and B. melitensis Rev.1 strains against a B. melitensis H38 challenge. Brucella counts in spleens of vaccinated mice are significantly different from those of the control mice: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data were obtained at week 2 () and week 8 () postchallenge.