Early detection of maedi-visna (ovine progressive pneumonia) virus seroconversion in field sheep samples.

The aim of this work was to investigate whether an enzyme-linked immunosorbent assay (ELISA) was useful for early detection of maedi-visna virus (MVV) infection in sheep under field conditions. An ELISA based on p25 recombinant protein and a gp46 synthetic peptide was used. Sequentially obtained serum samples (n = 1,941) were studied for 4 years. ELISA results were compared with those of the agar gel immunodiffusion (AGID) test, and results of both tests were compared with a reference result established using consensus scores for at least 2 of 3 serologic techniques (AGID, ELISA, and western blotting, which was used to resolve result discrepancies between the other 2 techniques). A total of 247 discrepancies were observed between ELISA and AGID. Of these, 131 were due to an earlier detection of 120 sera by the ELISA and 11 sera by AGID. The remaining discrepancies (116) were due to the presence of false reactions in both tests. Fewer false-negative results were found by ELISA than with AGID (6 vs. 69 sera, respectively), whereas the number of false-positive results was virtually the same for ELISA and AGID (21 vs. 20, respectively). In relation to the reference result, ELISA sensitivity and specificity were 97.8% and 98.2%, respectively, whereas values for AGID were 76.3% and 98.3%, respectively. The agreement between ELISA and the reference result was higher than that between AGID and the reference result (K value: 0.96 and 0.77, respectively). A variation in the ELISA signal (based on optical density) was observed during the study period, suggesting different antibody levels throughout the animal's life. The ELISA was useful for detecting MVV-infected sheep in field conditions and has potential for use in control and eradication programs.

Genetic diversity of protease and reverse transcriptase sequences in non-subtype-B human immunodeficiency virus type 1 strains: evidence of many minor drug resistance mutations in treatment-naive patients.

Most human immunodeficiency virus (HIV) drug susceptibility studies have involved subtype B strains. Little information on the impact of viral diversity on natural susceptibility to antiretroviral drugs has been reported. However, the prevalence of non-subtype-B (non-B) HIV type 1 (HIV-1) strains continues to increase in industrialized countries, and antiretroviral treatments have recently become available in certain developing countries where non-B subtypes predominate. We sequenced the protease and reverse transcriptase (RT) genes of 142 HIV-1 isolates from antiretroviral-naive patients: 4 belonged to group O and 138 belonged to group M (9 subtype A, 13 subtype B, 2 subtype C, 5 subtype D, 2 subtype F1, 9 subtype F2, 4 subtype G, 5 subtype J, 2 subtype K, 3 subtype CRF01-AE, 67 subtype CRF02-AG, and 17 unclassified isolates). No major mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors were detected. Major mutations linked to resistance to non-NRTI agents were detected in all group O isolates (A98G and Y181C) and in one subtype J virus (V108I). In contrast, many accessory mutations were found, especially in the protease gene. Only 5.6% of the 142 strains, all belonging to subtype B or D, had no mutations in the protease gene. Sixty percent had one mutation, 22.5% had two mutations, 9.8% had three mutations, and 2.1% (all group O strains) had four mutations. In order of decreasing frequency, the following mutations were identified in the protease gene: M36I (86.6%), L10I/V (26%), L63P (12.6%), K20M/R (11.2%), V77I (5.6%), A71V (2.8%), L33F (0.7%), and M46I (0.7%). R211K, an accessory mutation associated with NRTI resistance, was also observed in 43.6% of the samples. Phenotypic and clinical studies are now required to determine whether multidrug-resistant viruses emerge more rapidly during antiretroviral therapy when minor resistance-conferring mutations are present before treatment initiation.

Most env and gag subtype A HIV-1 viruses circulating in West and West Central Africa are similar to the prototype AG recombinant virus IBNG.

The genetic subtype was identified in gag and env of 219 HIV-1-positive samples collected in different African countries, 44 from Senegal, 55 from Cameroon, 82 from Gabon, and 38 from Djibouti. In total, 20 (9.1%) samples had discordant subtypes between gag and env, 6 of 44 (13.9%) in Senegal, 4 of 55 (7.2%) in Cameroon, 1 of 38 (2.6%) in Djibouti, and 10 of 82 (12.1%) in Gabon. Subtypes A and G were predominantly involved in the recombination events. Phylogenetic tree analysis of gag showed that an important number of the A sequences form a distinct subcluster with the AG-IBNG prototype strain (a complex A/G mosaic virus): 27 of 32 (84.3%) in Senegal, 12 of 17 (70.6%) in Nigeria, 24 of 39 (61.5%) in Cameroon, and 38 of 70 (54.3%) in Gabon. Full-length genome analysis of 3 and additional sequences in pol for 10 such strains confirmed that they have a similar complex A/G mosaic genomic structure. These data suggest that in West Africa, most probably between 60% and 84% of the subtype A viruses are recombinant AG-IBNG viruses. This finding has potential implications on future vaccine, diagnostic, and treatment strategies. The actual and future role of these viruses in the global pandemic must be monitored in all new molecular epidemiologic studies, a discrimination between subtype A and AG-IBNG-like viruses is necessary.

Near-full-length genome sequencing of divergent African HIV type 1 subtype F viruses leads to the identification of a new HIV type 1 subtype designated K.

We recently reported a high divergence among African subtype F strains. Three well-separated groups (F1, F2, and F3) have been shown based on the phylogenetic analysis of the p24 gag and envelope sequences with genetic distances similar to those observed for known subtypes. In this study, we characterized the near-full-length genomes of two strains from epidemiological unlinked individual belonging to each of the subgroups: F1 (96FR-MP411), F2 (95CM-MP255 and 95CM-MP257), and F3 (96CM-MP535 and 97ZR-EQTB11). Phylogenetic analysis of the near-full-length sequences and for each of the genes separately showed the same three groups, supported by high bootstrap values. Diversity plotting, BLAST subtyping, and bootstrap plotting confirmed that the divergent F strains correspond to nonrecombinant viruses. The divergence between F1 and F2 is consistently lower than that seen in any other intersubtype comparison, with the exception of subtypes B and D. Based on all the different analyses, we propose to divide subtype F into two subclades, with F1 gathering the known subtype F strains from Brazil and Finland, and our African strain (96FR-MP411), and F2 containing the 95CM-MP255 and 95CM-MP257 strains from Cameroon. The F3 strains, 97ZR-EQTB11 from the Democratic Republic of Congo and 96CM-MP535 from Cameroon, meet the criteria of a new subtype designated as K. The equidistance of subtype K to the other subtypes of HIV-1 suggests that this subtype existed as long as the others, the lower distance between B and D, and between F1 and F2 suggest a more recent subdivision for these latter strains.

DNA vaccination with genes encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice.

C57BL/6, C3H, and BALB/c mice were vaccinated with plasmids encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2, previously described as strong inducers of immunity. Seroconversion for the relevant antigen was obtained in the majority of the animals. T. gondii lysate stimulated specific T-cell proliferation and secretion of gamma interferon (IFN-gamma) in spleen cell cultures from vaccinated BALB/c and C3H mice but not in those from control mice. Although not proliferating, stimulated splenocytes from DNA-vaccinated C57BL/6 mice also produced IFN-gamma. No interleukin-4 was detected in the supernatants of lysate-stimulated splenocytes from DNA-vaccinated mice in any of the mouse strains evaluated. As in infected animals, a high ratio of specific immunoglobulin G2a (IgG2a) to IgG1 antibodies was found in DNA-vaccinated C3H mice, suggesting that a Th1-type response had been induced. For BALB/c mice, the isotype ratio of the antibody response to DNA vaccination was less polarized. The protective potential of DNA vaccination was demonstrated in C3H mice. C3H mice vaccinated with plasmid encoding GRA1, GRA7, or ROP2 were partially protected against a lethal oral challenge with cysts of two different T. gondii strains: survival rates increased from 10% in controls to at least 70% after vaccination in one case and from 50% to at least 90% in the other. In vaccinated C3H mice challenged with a nonlethal T. gondii dose, the number of brain cysts was significantly lower than in controls. DNA vaccination did not protect BALB/c or C57BL/6 mice. Our results demonstrate for the first time in an animal model a partially protective effect of DNA vaccination against T. gondii.

The identification of a complex A/G/I/J recombinant HIV type 1 virus in various West African countries.

In this sequence note we describe the full-length genome sequence of an HIV-1 isolate originating from the west African country of Mali. The phylogenetic tree analysis from the near full-length genome shows that the 95ML84 strain forms a separate cluster, supported by 100% of the bootstrap values, with the previously described A/G/J/? mosaic virus BFP90 from Burkina Faso. Additional analysis showed that throughout the genome the lowest diversity was seen between the 95ML84 and the BFP90 viruses, and bootscan analysis showed a similar complex genomic structure. In addition to the initial report describing the BFP90 virus as an A/G/J/? recombinant, our data show that for the BFP90 and 95ML84 strains the unclassified region corresponds to subtype I. The A/G/I/J BFP90 and 95ML84 strains represent the fifth and most complex circulating recombinant form of HIV-1 detected so far, and our data show its presence in various West African countries. Subtype I and J sequences, initially considered rare, seem to have broadened their geographical spread by way of these recombinant forms.

In vivo expression and distribution of dense granule protein 7 (GRA7) in the exoenteric (tachyzoite, bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii.

The in vivo expression and distribution of the dense granule protein GRA7 was examined in both the exoenteric (tachyzoite and bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii by immunocytochemistry. There was strong staining of GRA7 in granules within all the infectious stages (tachyzoite, bradyzoite, merozoite and sporozoite). During tachyzoite development, GRA7 was secreted and was associated with the parasitophorous vacuole. In contrast, although there was staining of granules within the bradyzoites of more mature cysts, there appeared to be little staining of the tissue cyst wall or host cell. The apparent stage-specific variation in secretion of GRA7 between tachyzoites and bradyzoites was confirmed by double labelling using stage-specific markers (SAG1 and BAG1). In the enteric forms in the cat gut there was strong labelling of the PV containing early asexual and sexual stages and staining of a few granules in the apical cytoplasm of the merozoite. The positive enteric staining pattern differentiates GRA7 from the other GRA proteins (GRA1-6) which were absent in the merozoites and enteric stages. The staining pattern of GRA7 with strong staining during tachyzoite and enteric development and reduced staining in the tissue cysts is similar to that seen for NTPases. The function of GRA7 is unknown but it is unique among the dense granule proteins in being expressed in all the infectious forms of T. gondii which would point to a basic role in the vacuolar adaptations required for active parasite development.

A new sensitive serological assay for detection of lentivirus infections in small ruminants.

Lentivirus infections in small ruminants represent an economic problem affecting several European countries with important sheep-breeding industries. Programs for control and eradication of these infections are being initiated and require reliable screening assays. This communication describes the construction and evaluation of a new serological screening enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (MVV) in sheep and to caprine arthritis encephalitis virus (CAEV) in goats. The solid phase is sensitized with a combination of the major core protein p25 of MVV produced in Escherichia coli and a peptide derived from the immunodominant region of the viral transmembrane protein gp46. The peptide carries an N-terminal biotin residue and is complexed with streptavidin prior to being coated. The new assay was evaluated with 2,336 sheep serum samples from different European countries with large differences in the levels of prevalence of MVV infections, and the results have been compared to those of the standard agar gel immunodiffusion test. Discrepant samples were analyzed by Western blotting with viral lysate, and most sera could be classified unambiguously. The estimated overall sensitivity of the new ELISA was 99.4% (95% confidence interval [CI], 98.4 to 99. 8%) and the specificity was 99.3% (95% CI, 98.7 to 99.6%). A limited set of goat sera (n = 212) was also analyzed, with similar results. These data indicate that the new assay is a reliable tool that can be used in control and eradication programs for small ruminant lentivirus infections.

Characterization of a highly replicative intergroup M/O human immunodeficiency virus type 1 recombinant isolated from a Cameroonian patient.

A Cameroonian patient with antibodies reacting simultaneously to human immunodeficiency virus type 1 (HIV-1) group O- and group M-specific V3-loop peptides was identified. In order to confirm that this patient was coinfected with both viruses, PCRs with O- and M-specific discriminating primers corresponding to different regions of the genome were carried out with both primary lymphocyte DNA and the corresponding viral strains isolated from three consecutive patient samples. The PCR data suggested that this patient is coinfected with a group M virus and a recombinant M/O virus. Indeed, only type M gag sequences could be amplified, while for the env region, both type M and O sequences were amplified, from plasma or from DNA extracted from primary lymphocytes. Sequence analysis of a complete recombinant genome isolated from the second sample (97CA-MP645 virus isolate) revealed two intergroup breakpoints, one in the vpr gene and the second in the long terminal repeat region around the TATA box. Comparison of the type M sequences shared by the group M and the recombinant M/O viruses showed that these sequences were closely related, with only 3% genetic distance, suggesting that the M virus was one of the parental viruses. In this report we describe for the first time a recombination event in vivo between viruses belonging to two different groups, leading to a replicative virus. Recombination between strains with such distant lineages (65% overall homology) may contribute substantially to the emergence of new HIV-1 variants. We documented that this virus replicates well and became predominant in vitro. At this time, group O viruses represent a minority of the strains responsible for the HIV-1 pandemic. If such recombinant intergroup viruses gained better fitness, inducing changes in their biological properties compared to the parental group O virus, the prevalences of group O sequences could increase rapidly. This will have important implications for diagnosis of HIV-1 infections by serological and molecular tests, as well as for antiviral treatment.

Immunogenicity of recombinant BCG producing the GRA1 antigen from Toxoplasma gondii.

Toxoplasmosis is a major parasitic disease, responsible for foetopathy in humans and domestic animals, especially sheep. Toxoplasma gondii infection generally protects immunocompetent hosts against subsequent reinfection, suggesting that efficacious vaccines can be developed against this disease. Excreted/secreted T. gondii antigens have previously been shown to provide immunoprotection in small rodents, and protective immunity is thought to be cell-mediated. Mycobacterium bovis BCG is known to be a good inducer of cellular immunity. In this study, we have developed a BCG strain which produces and secretes GRA1, one of the major excreted/secreted T. gondii antigens. This strain does not carry antibiotic-resistance determinants and is therefore safe for the environment. The intraperitoneal immunisation of OF1 outbred mice with this BCG strain failed to induce GRA1-specific humoral or cellular immune responses and only conferred a very limited degree of protection against challenge with virulent T. gondii. However, in sheep immunised subcutaneously and boosted intravenously, this recombinant BCG strain induced GRA1-specific cell-mediated responses, as evidenced by the proliferation of peripheral blood mononuclear cells and by the production of IFN-gamma, although it failed to elicit GRA1-specific antibody responses. Following oocyst challenge infection, sheep immunised with recombinant BCG exhibited an abbreviated temperature response compared with controls, suggesting partial protection.

Human cyclophilin has a significantly higher affinity for HIV-1 recombinant p55 than p24.

The ability of cyclophilin to bind a panel of recombinant HIV-gag proteins was assessed using sensitive, quantitative, sandwich enzyme-linked immunosorbant assays (ELISAs). Significantly higher binding to cyclophilin was observed when recombinants contained at least 12 carboxy-terminal amino acids of p17 in addition to p24 sequences. These results indicate that the carboxy-terminus of p17 is important for optimal binding of cyclophilin to p24 and support the theory that cyclophilin acts on the uncleaved HIV-1 gag (p17-p24) precursor.

Evaluation of recombinant dense granule antigen 7 (GRA7) of Toxoplasma gondii for detection of immunoglobulin G antibodies and analysis of a major antigenic domain.

Dense granule protein 7 (GRA7) of Toxoplasma gondii was expressed in Escherichia coli as a fusion protein. The leader peptide contained a 25-amino-acid mouse tumor necrosis factor fragment and six histidyl residues. After purification by metal chelate affinity chromatography, the antigen was evaluated in an enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG). For two sets of IgG-positive human serum samples, obtained from routine screening, an overall sensitivity of 81% was obtained. For chronic-phase sera, the sensitivity of detection was 79%, but chronic-phase sera with low titers were more difficult to detect (65% sensitivity for sera with immunofluorescence titer of 1/64). When GRA7 was combined with Tg34AR (rhoptry protein 2 C-terminal fragment), the sensitivity rose to 96%. For a set of acute-phase serum samples tested on GRA7, the sensitivity of detection was 94%, and high-titer IgM-positive sera were detected at an especially high rate. In contrast, when Tg34AR was used, the sensitivity was only 85% for this latter set of serum samples. Three truncated GRA7 fragments containing the same leader peptide as that of recombinant GRA7 were produced. The shortest fragment (97 N-terminal amino acids) was not reactive with human sera or with a specific anti-GRA7 monoclonal antibody, while the two larger fragments were reactive. The most important antigenic domain of GRA7 for human sera was localized between residues 97 and 146. The epitope for the specific monoclonal antibody could be further narrowed down by the use of synthetic peptides, but this epitope is not recognized by sera from T. gondii-infected humans. These results indicate that GRA7 may be considered as an additional tool for studying the immune response to T. gondii.

Genetic characterization of accessory genes from human immunodeficiency virus type 1 group O strains.

Human immunodeficiency virus type 1 (HIV-1) group O strains have been described as highly divergent, compared with the vast majority of the viruses involved in the worldwide AIDS pandemic, classified in group M. To gain new insights into the diversity and genetic characteristics of group O, we have sequenced the accessory gene region (from vif to vpu) of 14 isolates. Analyses of the deduced amino acid sequences for Vif, Vpr, the first exon of Tat, and Vpu indicate that most of the functional domains of these proteins, as described for group M viruses, are highly conserved and retained among all the group O strains we have characterized. The only difference concerns the Vif phosphorylation sites, which are absent in all of the group O isolates we have sequenced; in contrast, they are well conserved in nearly all of the group M isolates, in which they play critical roles in the regulation of viral replication and infectivity. As already observed for group M isolates, the Vpu protein is also highly diverse among group O strains. Phylogenetic analyses of these sequences indicate that HIV-1 group O can be separated into four different clusters, containing most of the strains we have characterized (except one, which clusters outside of the analyzed viruses). Taking into account the criteria used for clades in group M, we were not able to define group O clades definitively.

Differential diagnosis of HIV type 1 group O and M infection by polymerase chain reaction and PstI restriction analysis of the pol gene fragment.

HIV-1 group O serological screening or confirmation strategies so far have not proved 100% sensitive and specific, indicating a lack of antibody reactivity or cross-reactivity with group O antigens. Therefore, genetic analysis currently represents the only method by which confirm presumed HIV-1 group O or group O/M infections. We have optimized the sensitivity (100%) and specificity (100%) of an HIV-1 group O/M-specific PCR of a pol gene fragment. In addition, we report on a highly sensitive (97.2%) and specific (100%) method for differentiation between HIV-1 group O and group M viruses, using PCR and PstI enzyme restriction fragment analysis of a pol fragment. Compared with sequencing, these methods are fast, inexpensive, and simple.

Identification and heterologous expression of a new dense granule protein (GRA7) from Toxoplasma gondii.

Immunoscreening of an expression library constructed with Toxoplasma gondii tachyzoite mRNA with sera from toxoplasmosis-positive humans has led to the identification of a new parasite antigen. Sequence analysis of the gene encoding this antigen allowed the calculation of the theoretical molecular mass (25,857 Da) and showed that the protein contains a putative signal sequence. The C-terminal region contains two hydrophobic regions, the last of which has the characteristics of a membrane-spanning domain. When the protein was heterologously expressed in E. coli and tested by Western blot, it reacted with the human sera originally used for screening. The new antigen also reacted with a monoclonal antibody raised against the entire parasite. Ultrastructural analysis showed that the protein is localized in the dense granules. After host cell invasion, the protein is secreted into the vacuolar network, the parasitophorous vacuole membrane, and into extensions protruding in the cytoplasm. Therefore, it is suggested to designate this new dense granule protein GRA7, following the established nomenclature for this protein family.

Vaccination with plasmid DNA encoding mycobacterial antigen 85A stimulates a CD4+ and CD8+ T-cell epitopic repertoire broader than that stimulated by Mycobacterium tuberculosis H37Rv infection.

Vaccination of mice with plasmid DNA carrying the gene for the major secreted mycobacterial antigen 85A (Ag85A) from Mycobacterium tuberculosis is a powerful technique for generating robust specific Thl helper T-cell responses, CD8+-mediated cytotoxicity, and protection against M. tuberculosis challenge (K. Huygen et al., Nat. Med. 2:893-898, 1996). We have now analyzed in more detail the antigen-specific immune CD4+- and CD8+-T-cell responses induced in BALB/c mice vaccinated with Ag85A DNA and have compared these responses to those generated by intravenous infection with M. tuberculosis. T-cell-epitope mapping, as measured by interleukin-2 and gamma interferon secretion from splenic T cells restimulated in vitro with synthetic 20-mer peptides spanning the complete mature sequence of Ag85A, demonstrated that DNA vaccination stimulated a stronger and broader T-cell response than did M. tuberculosis infection. Moreover, elevated cytotoxic T lymphocyte (CTL) activity against Ag85A-transfected and peptide-pulsed P815 target cells could be generated exclusively by vaccination with plasmid DNA, not following M. tuberculosis infection. By using DNA vaccination, three Ag85A CTL epitopes with predicted major histocompatibility complex class I binding motifs were defined. One of them was previously reported as a dominant, promiscuously recognized T-cell epitope in healthy humans with primary infections. These data strengthen the potential of DNA vaccination with respect to inducing antituberculous immunity in humans.

Assessment of a new immunoassay for serological confirmation and discrimination of human T-cell lymphotropic virus infections.

The present study evaluated a new confirmatory assay for antibodies to human T-cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) proteins performed with serum samples from various commercial sources. The new test is a line immunoassay (LIA) with a nylon membrane sensitized with the most relevant antigens of HTLVs: the envelope gp46 and gp21 as well as the gag p24 and p19 antigens, represented by either recombinant proteins or synthetic peptides. A total of 176 serum or plasma samples were tested, of which 66 were HTLV-1 positive, 72 were HTLV-2 positive, and 38 were HTLV negative; of the 38 HTLV-negative samples 23 were indeterminate by Western blotting (WB). Serially diluted samples (n = 33) from HTLV-1- and HTLV-2-infected patients were also analyzed to determine the sensitivity of the new assay. The new confirmatory assay (INNO-LIA HTLV) performed markedly better than WB assays for those samples reactive by screening. Accurate confirmation of the presence of HTLV-1 and HTLV-2 antibodies and accurate discrimination of HTLV-1 and HTLV-2 antibodies were obtained for all the HTLV-seropositive samples. Due to its enhanced specificity and sensitivity, the new assay not only improves the ability to confirm and discriminate HTLV infections but also eliminates the vast majority of WB-indeterminate and false-positive specimens.

Humoral immune responses of Brucella-infected cattle, sheep, and goats to eight purified recombinant Brucella proteins in an indirect enzyme-linked immunosorbent assay.

Brucellosis research is currently focused on the identification of nonlipopolysaccharide (LPS) antigens which could potentially be useful for the specific serologic diagnosis of brucellosis as well as for vaccinal prophylaxis. On the basis of previous reports, we selected eight Brucella proteins (OMP36, OMP25, OMP19, OMP16, OMP10, p17, p15, and p39) as candidate antigens to be further evaluated. The genes encoding these proteins were cloned, sequenced, and overexpressed in Escherichia coli. The recombinant proteins were purified with a polyhistidine tag and metal chelate affinity chromatography and evaluated in an indirect enzyme-linked immunosorbent assay (iELISA). The specificity of the iELISA was determined with sera from healthy cattle, sheep, and goats and ranged from 95 to 99%, depending on the recombinant antigen and the species tested. Sera from experimentally infected, and from naturally infected, animals were used to evaluate the sensitivity of the iELISA. The antiprotein antibody response was often delayed when compared to the anti-smooth LPS (S-LPS) response and was limited to animals which developed an active brucellosis infection (experimentally infected pregnant animals and sheep and goats from areas where brucellosis is still endemic). Among the recombinant antigens, the three cytoplasmic proteins (p17, p15, and p39) gave the most useful results. More than 80% of the animals positive in S-LPS serology were also positive with one of these cytoplasmic proteins alone or a combination of two of them. None of the recombinant antigens detected experimentally infected nonpregnant cows and sheep or naturally infected cattle. This study is a first step towards the development of a multiprotein diagnostic reagent for brucellosis.