Brucella spp. 234 The mechanism of Brucella pathogenesis and infection of natural animal hosts is not completely understood (Corbel, 1997). It is well accepted that its pathogenesis relies on its ability to live and replicate inside the vacuolar phagocytic compartment of macrophages (Kohler et al., 2002). The host macrophage-Brucella interaction is critical for establishment of chronic Brucella infections. The Type IV secretion system encoded by the virB operon and the two-component regulatory system encoded by the bvrRS operon are required for successful replication of Brucella in macrophages. Smooth Brucella strains with intact lipopolysaccharide (LPS) O-side chains are virulent and invade macrophages through lipid rafts. On entry into the macrophage, Brucella reside in an acidified compartment that fuses with components of the early endosomal pathway. The majority of Brucella die at the early infection stage. A subpopulation of virulent Brucella strains is trafficked to an intracellular compartment known as the replicative phagosome (brucellosome) through continual interactions between the Brucella-containing vacuoles and the endoplasmic reticulum of the host macrophages. Once inside the replicative phagosome, Brucella are resistant to further attack and rapidly propagate. Virulent Brucella also inhibit macrophage apoptosis that in turn favors pathogen survival and replication. Brucella also infects placental trophoblast cells where acute infection leads to abortion and disease transmission (Roop et al., 2004). Brucellosis Both humoral and cell-mediated immune responses can influence the course of a Brucella infection, cell-mediated immune responses are essential for clearance of intracellular Brucella (Schurig et al., 2002). Brucella has a broad host range infecting vertebrate animal species and humans. Brucella infections are most often identified in humans, cattle, swine, goats, sheep, deer, caribou, elk, dogs and coyotes. There are six Brucella species based on the preferential host specificity: B. melitensis (goats), B. abortus (cattle), B. suis (swine), B. canis (dogs), B. ovis (sheep) and B. neotomae (desert mice). The first four species are pathogenic and exhibit a decreasing order of severity in humans. Therefore, brucellosis is a zoonotic disease (Corbel, 1997). Mouse models are often used in Brucella vaccine research. BALB/c and C57BL/10 mice differ in their abilities to induce immune responses and protection against virulent Brucella infections (Fernandes et al., 1996) (Li, 1991) (Montaraz et al., 1986). Brucella species are gram-negative, facultative intracellular bacteria that cause brucellosis (or called Malta fever, undulant fever, Bang's disease, Mediterranean fever ) in humans and a variety of animals (Corbel, 1997). B. melitensis, B. abortus, B. suis, and B. canis are pathogenic to humans. The first three species have been identified as priority agents amenable for use in biological warfare and bio-terrorism, and are listed as NIAID and CDC category B priority pathogens. To date, there is no safe, effective Brucella vaccine available for human use. The vaccine strains used in animals are considered to be too virulent and unsafe for humans (Schurig et al., 1991). Novel vaccines that are non-infectious to humans but effective in stimulating a broad protective immune response are needed. Baboon Papio cynocephalus 9556 Bank vole Clethrionomys glareolus 447135 Bear Ursus americanus 9643 Birds Passeroidea 175121 Brown Trout Salmo trutta 8032 Buffalo Bison bison 9901 Carnivores Vulpes 9625 Cat Felis catus 9685 Catfishes Siluriformes 7995 Cattle Bos taurus 9913 Chicken Gallus gallus 9031 Chimpanzee Pan troglodytes 9598 chinchillas Chinchillidae 10150 Copper Pheasant Syrmaticus soemmerringii 9067 Deer Cervus elaphus 9860 Deer mouse Peromyscus maniculatus 10042 Dog Canis familiaris 9615 Ducks Anas 8835 Ferret Mustela putorius furo 9669 Fish Hyperotreti 117565 Gerbil Gerbillina 10045 Goat Capra hircus 9925 Gray wolf Canis lupus 9612 Guinea pig Cavia porcellus 10141 Hamster Mesocricetus auratus 10036 Horse Equus caballus 9796 Human Homo sapiens 9606 Macaque Macaca fascicularis 9541 Mongolian Gerbil Meriones unguiculatus 10047 Monkey Platyrrhini 9479 Mouse Mus musculus 10090 None None Parrot Psittacidae 9224 Pig Sus scrofa 9823 Rabbit Oryctolagus cuniculus 9986 Rainbow trout Oncorhynchus mykiss 8022 Rat Rattus 10114 Raven Corvus corax 56781 sei whale Balaenoptera borealis 9768 Sheep Ovis aries 9940 Squirrel Spermophilus richardsonii 37591 Tree shrew Tupaiidae 9393 Trouts, salmons & chars Salmoninae 504568 Turkey Meleagris gallopavo 9103 Vole Microtus ochrogaster 79684 Water buffalo Bubalus bubalis 391902 B. abortus and B. melitensis mutants with unmarked deletion of asp24, virB2, or manBA VO_0002810 Aerosal Vaccine Each vaccine consisted of 5 × 10^7 CFU/ml in a chamber nebulizer, which is thousands of organisms per dose. The bacteria were introduced in Farrell's medium: TSA supplemented with 5 mg/liter nalidixic acid, 25,000 IU/liter bacitracin, 100 mg/liter cycloheximide, 5000 IU/liter polymyxin B sulfate, 20 mg/liter vancomycin, 100,000 IU/liter nystatin 10% (vol/vol) horse serum, and 2% (wt./vol.) dextrose (Kahl-McDonagh et al., 2007). The vaccines tested in this study include B. abortus unmarked mutants BAΔasp24, BAΔvirB2, and BAΔmanBA, and B. melitensis unmarked mutants BMΔasp24, BMΔvirB2, and BMΔmanBA (Kahl-McDonagh et al., 2007). Gene mutation This asp24 mutant is from Brucella abortus (Kahl-McDonagh et al., 2007). Gene mutation This virB2 mutant is from Brucella melitensis (Kahl-McDonagh et al., 2007). Gene mutation This manBA mutant is from Brucella melitensis (Kahl-McDonagh et al., 2007). Gene mutation This manBA mutant is from Brucella melitensis (Kahl-McDonagh et al., 2007). BALB/c Groups of four or five female 6- to 8-week-old BALB/c mice were vaccinated via i.p. injection of 1 × 10^6 CFU/ml of unmarked deletion mutant or PBS for naïve controls (Kahl-McDonagh et al., 2007). Mice receiving a dose of 5 × 10^7 CFU/ml had lung colonization with B. abortus strain 2308 that gradually increased over the first 4 weeks postchallenge, then gradually decreased over the following 4 weeks to 90% of the maximum value. Despite this slight decrease, colonization by the organism in the other tissues was consistent with a chronic infection. Colonization of the liver, although barely detectable at 1 week postchallenge, steadily increased over the first 4 weeks postchallenge and then declined negligibly between weeks 4 and 8. The spleens of infected mice displayed a colonization pattern similar to that of the livers, although the total number of CFU recovered was consistently higher. Spleen colonization increased between weeks 4 and 8, consistent with a persistent infection (Kahl-McDonagh et al., 2007). The bacterial burden in the spleen following aerosol challenge was reduced 2.02 U by vaccination with BAΔasp24 and by 0.86 U by vaccination with BAΔvirB2 relative to the burden in naïve mice. The rough BAΔmanBA mutant was unable to elicit significant protective immunity relative to the immunity of naïve mice. At 24 weeks postvaccination, less protection was observed in the lungs of mice vaccinated with BAΔasp24 and BAΔvirB2, although the protection was significantly greater than that observed for naïve controls. The rough mutant, BAΔmanBA, protected mouse lungs to a lesser degree, which was not significant(Kahl-McDonagh et al., 2007) . Mice receiving a dose of 5 × 10^7 CFU/ml had lung colonization with 2308 that gradually increased over the first 4 weeks postchallenge, then gradually decreased over the following 4 weeks to 90% of the maximum value. Despite this slight decrease, colonization by the organism in the other tissues was consistent with a chronic infection. Colonization of the liver, although barely detectable at 1 week postchallenge, steadily increased over the first 4 weeks postchallenge and then declined negligibly between weeks 4 and 8. The spleens of infected mice displayed a colonization pattern similar to that of the livers, although the total number of CFU recovered was consistently higher. Spleen colonization increased between weeks 4 and 8, consistent with a persistent infection (Kahl-McDonagh et al., 2007). BALB/c mice were challenged with an aerosol chamber dose of 5 × 10^9 CFU/ml of the homologous wild-type strain at 20 weeks postvaccination. Four weeks after the virulent challenge (corresponding to 24 weeks postvaccination), the mice were euthanized and the recovery of the challenge orgainsm was measured (Kahl-McDonagh et al., 2007). B. abortus DNA vaccine encoding RplL and Omp16 VO_0011380 DNA vaccine Research Expression vector pcDNA3.1 Intramuscular injection (i.m.) Intramuscular injection (i.m.) B. abortus ribosomal protein rplL and outer membrane lipoprotein 16 DNA vaccine construction Full-length open reading frames of the L7/L12 gene and Omp16 gene were amplified with PCR from the genome of attenuated B. abortus strain RB51. The gene amplified with L7/L12 primers (FL and RL-1) and the gene amplified with Omp16 primers (FO and RO) were inserted into pcDNA3.1(+) vector (Invitrogen) at the EcoRV/XhoI and BamHI/XhoI sites to construct recombinant plasmids L7/L12-pcDNA3.1 and Omp16-pcDNA3.1, respectively. To construct the recombinant fusion plasmid L7/L12-Omp16-pcDNA3.1, the L7/L16 gene fragment was amplified with the L7/L16 PCR primers (FL and RL-2) first, which removed only the TAA stop codon from the L7/L16 gene. This PCR product and the amplified Omp16 gene above were digested with EcoRV/BamHI and BamHI/XhoI, respectively, and ligated with T4 ligase; the ligated product was then inserted into the pcDNA3.1(+) vector between the EcoRV and XhoI sites (Luo et al., 2006b). DNA vaccine construction Full-length open reading frames of the L7/L12 gene and Omp16 gene were amplified with PCR from the genome of attenuated B. abortus strain RB51. The PCR primers were designed as shown in Table 1. The gene amplified with L7/L12 primers (FL and RL-1) and the gene amplified with Omp16 primers (FO and RO) were inserted into pcDNA3.1(+) vector (Invitrogen) at the EcoRV/XhoI and BamHI/XhoI sites to construct recombinant plasmids L7/L12-pcDNA3.1 and Omp16-pcDNA3.1, respectively. To construct the recombinant fusion plasmid L7/L12-Omp16-pcDNA3.1, the L7/L16 gene fragment was amplified with the L7/L16 PCR primers (FL and RL-2) first, which removed only the TAA stop codon from the L7/L16 gene. This PCR product and the amplified Omp16 gene above were digested with EcoRV/BamHI and BamHI/XhoI, respectively, and ligated with T4 ligase; the ligated product was then inserted into the pcDNA3.1(+) vector between the EcoRV and XhoI sites (Luo et al., 2006b). BALB/c The mice were anesthetized with methoxyflurane (Metofan; Mallinckrodt) and inoculated intramuscularly with 100 μg of pcDNA3.1-L7/L12-Omp16, pcDNA3.1-L7/L12, or pcDNA3.1-Omp16 in 100 μl of PBS (50 μl of the solution was injected into each tibialis anterior muscle). The control mice were infected with PBS or the expression vector alone (pcDNA3.1). Each mouse in another group was injected with 10 μg of rL7/L12-Omp16 in 100 μl PBS according to the same schedule. Each mouse was injected on weeks 0, 2, and 4. The mice used as positive controls were inoculated intraperitoneally on day 0 with 2× 10^8 CFU of B. abortus strain RB51 in 0.2 ml of PBS (Luo et al., 2006b). This divalent DNA vaccine induced a significant level of protection against challenge with the virulent B. abortus in BALB/c mice (Luo et al., 2006b). Two weeks after the final vaccination, five mice from each group were challenged intraperitoneally with relatively higher dose of strain 544 (5 × 10^5 CFU). Four weeks postchallenge, the mice were killed by cervical dislocation, and their spleens were removed aseptically and weighed (Luo et al., 2006b). Sera collected 2 weeks after the last immunization were assayed for the presence of L7/L12- and/or Omp16-specific antibodies by ELISA. The pcDNA 3.1-L7/L12-OMP16 vaccine elicited significantly higher levels of IgG1 antibodies than did the negative control vaccine, pcDNA 3.1 (Luo et al., 2006b). Sera collected 2 weeks after the last immunization were assayed for the presence of L7/L12- and/or Omp16-specific antibodies by ELISA. The pcDNA 3.1-L7/L12-OMP16 vaccine elicited significantly higher levels of IgG2a antibodies than did the negative control vaccine, pcDNA 3.1 (Luo et al., 2006b). B. abortus DNA vaccine expressing BCSP31, SOD and L7/L12 VO_0000321 DNA vaccine pJW4303 [Ref480:Yu et al., 2007] Vaccine combines three known antigens, BCSP31, SOD, and L7/L12, in a single DNA vaccine. None The coding regions for the antigens BCSP31, SOD, and L7/L12 were amplified from B. abortus strain 2308 chromosomal DNA. GenBank accession numbers for the sequences reported in this study are M20404 for BCSP31, L19101 for L7/L12, and AE017334 for SOD. All antigen-coding regions were fused individually to the tissue plasminogen activator signal sequences, and the DNA constructs were purified using the Qiagen Mega plasmid DNA kit (Qiagen, Valencia, CA) and verified by commercial DNA sequencing. The DNA vaccine was diluted in saline solution to a final concentration of 1–2 mg/mL before use (Yu et al., 2007). B. abortus BCSP31, SOD, and L7/L12 (Yu et al., 2007). DNA vaccine construction The DNA vaccine comprising genes encoding the antigens BCSP31, superoxide dismutase (SOD), and L7/L12 was constructed and evaluated for its immunogenicity and protective efficacy (Yu et al., 2007). DNA vaccine construction DNA vaccine construction Mice vaccinated with the combined DNA produced a rapid and specific IgG response 3 weeks after the initial vaccination, and peak titers were detected 3 weeks after the last immunization. The S19-vaccinated mice also induced antibody production, but the rise was not as pronounced. Combined DNA–vaccinated mice had significantly higher BCSP31-, SOD-, L7/L12-, and Brucella-specific total IgG production than S19-vaccinated mice. Combined DNA–vaccinated mice produced significantly higher levels of IFN-g and TNF-a ( p < 0.01) compared with the two negative control groups. A significant T cell response was observed after incubating spleen cells from combined DNA–vaccinated mice with the specific antigens rBCSP31, rSOD, rL7/L12, and heat-killed B. abortus (Yu et al., 2007). Plasmids carrying BCSP31, SOD, and L7/L12 genes were administered by intramuscular injection of 150 ug DNA (50 ug of each plasmid) in 150 uL saline solution into each of the quadriceps. For the vector control group, 150 mg pJW4303 empty vector DNA in 150 mL saline was used for injection. As an additional negative control, mice were injected with 150 mL saline. Immunization of mice was repeated thrice at 3-week intervals. The positive control group of mice was vaccinated intraperitoneally with 5x10^6 colony-forming units (CFU) of B. abortus The levels of infection were evaluated by measuring CFU in the spleen. The combined DNA–vaccinated mice displayed a significantly higher level of protection than mice vaccinated with vector DNA or saline (3.58 log units higher; p < 0.001; n=10). In addition, our vaccine provided significantly higher protection than S19 (log units=2.87 for S19; p=0.034); that is, after infection, the number of CFU in the spleen of the combined DNA vaccine group was reduced to less than 20% of that in the S19 group. Indicating that the combined DNA vaccine affords a significant degree of protection against Brucella infection (Yu et al., 2007). Mice were challenged intravenously with 5x10^6 CFU (Yu et al., 2007). B. abortus DNA vaccine pcDNA-SOD VO_0000018 DNA vaccine pcDNA3 [Ref220:Munoz-Montesino et al., 2004] Cu-Zn superoxide dismutase (SOD) is one of the protective immunogens of Brucella abortus. Intramuscular injection with a plasmid DNA carrying the SOD gene (pcDNA-SOD) was able to induce a protective immune response in mice (Munoz-Montesino et al., 2004). Recombinant plasmid pBAII-3, containing the gene for B. abortus Cu-Zn SOD (sodC), and its own promoter, was initially obtained from a pUC9 genomic library of B. abortus strain 2308. A 1.1-kb fragment containing the sodC gene and its promoter sequences was excised and ligated into the expression vector pcDNA3 downstream of the cytomegalovirus promoter. The resulting plasmid was designated pcDNA-SOD. A colony of E. coli containing pcDNASOD was cultured and used for large-scale plasmid DNA isolation. The DNA was resuspended in PBS at a final concentration of 1 mg/ml. The pcDNA-SOD plasmid construct was verified by restriction digestion and by sequencing of the complete insert (Munoz-Montesino et al., 2004). B. abortus Cu/Zn Superoxide dismutase (Munoz-Montesino et al., 2004). DNA vaccine preparation B. abortus sodC was subcloned into the expression vector pcDNA3 (Munoz-Montesino et al., 2004). Animals vaccinated with pcDNA-SOD did not develop SOD-specific antibodies, at least until week 4 after immunization (the end of the experiment), and in vitro stimulation of their splenocytes with either recombinant Cu-Zn SOD or crude Brucella protein induced the secretion of gamma interferon (IFN-γ), but not interleukin-4, and elicited the induction of cytotoxic-T-lymphocyte activity. Upon analyzing the SOD-specific T-cell responses, the pcDNA-SOD vaccination was found to be stimulating both CD4+- and CD8+-T-cell populations. However, only the CD4+ population was able to produce IFN-γ and only the CD8+ population was able to induce cytotoxic activity (Munoz-Montesino et al., 2004). BALB/c mice Mice premedicated with atropine (16 µg/mouse) were anesthetized with ketamine (2 µg/mouse), their abdominal regions shaved. One 3-mm-long incision was made through the skin. The mice were injected in the spleen with 10 µg of plasmid DNA in 30 µl of PBS. Mice were injected i.m. in the right tibialis anterior muscle with 10 µg of plasmid DNA per mouse. The mice were vaccinated once with pcDNA-SOD construct or with pcDNA3 as a negative control. For protection assays, an additional group was inoculated with PBS as a negative control (Munoz-Montesino et al., 2004). Immunization (i.s.) with pcDNA-SOD induced a significant degree of protection (1.52-log-unit increase in protection) compared to the control group (P < 0.008). The mice that were immunized (i.m.) with pcDNA-SOD showed minimal protection, (P < 0.2). No significant difference was seen between the CFU numbers in control groups injected with pcDNA3 and PBS. Vaccination with pcDNA-SOD vaccination (i.s.) therefore provided a significant degree of protection against Brucella infection (Munoz-Montesino et al., 2004). Four weeks after vaccination, six mice from each group were challenged by intraperitoneal injection of 10^4 CFU of B. abortus 2308. Two weeks later, the infected mice were sacrificed, their spleens homogenized, and dilutions of the extract were plated to determine the number of Brucella CFU per spleen (Munoz-Montesino et al., 2004). pcDNA-SOD induced Cu-Zn SOD specific IFN-γ production from CD4+ but not CD8+ T cells from in pcDNA-SOD immunized mice (Munoz-Montesino et al., 2004). pcDNA-SOD did not induce interleukin-4 production in immunized mice (Munoz-Montesino et al., 2004). B. abortus DNA vaccine pcDNA3-SOD encoding Cu-Zn SOD VO_0004393 DNA vaccine Research pcDNA3 [Ref564:Onate et al., 2003] Intramuscular injection (i.m.) Intramuscular injection (i.m.) DNA vaccine construction Vector pcDNA3 expressed Cu-Zn SOD (Onate et al., 2003). Vaccination was able to induce a broad range of immune responses including antibody production, CD8+ cytotoxic T cells (CTLs), and CD4+ T helper cell activation (Onate et al., 2003). VO_0000286 Immunization with pcDNA-SOD resulted in a significantly higher degree of protection (2.16 log increase in protection) compared to the unimmunized control groups (P < 0.0005). The level of protection was similar to the one induced by B. abortus vaccine strain RB51. Thus, pcDNA-SOD vaccine afforded a significant degree of protection against Brucella infection (Onate et al., 2003). B. abortus DNA vaccine pcDNA3.1-p39-L7/L12 encoding p39-L7/L12 fusion protein VO_0004392 DNA vaccine Research pcDNA3.1 [Ref491:Luo et al., 2006a] Intramuscular injection (i.m.) Intramuscular injection (i.m.) p39-L7/L12 fusion protein from RB51 (Luo et al., 2006a) DNA vaccine construction DNA vaccine construction Inoculation of plasmid DNA containing the L7/L12-P39 gene leads to both TH1 type antibody and CMI responses (Luo et al., 2006a). VO_0000286 Among the three DNA vaccines (including pcDNA3.1(+)-L7/L12 and pcDNA3.1(+)-P39), the pcDNA3.1 (+)-L7/L12-P39 DNA vaccine could provide the highest protective level against Brucella infection, as it confers protection against A544 challenge (Luo et al., 2006a). B. abortus DNA vaccine pVF278 VO_0004546 DNA vaccine Research pVF [Ref2664:Sislema-Egas et al., 2012] Intramuscular injection (i.m.) Intramuscular injection (i.m.) DNA vaccine construction Mice injected with pVF278 had a dominant immunoglobulin G2a (IgG2a) response and elicited a T-cell-proliferative response (Sislema-Egas et al., 2012). VO_0003057 Data from two independent protection experiments demonstrated that immunization with pVF278 DNA vaccine resulted in a significantly degree of protection (1.28 log increase in protection) with P < 0.05 after determination of CFU in the spleen (Sislema-Egas et al., 2012). B. abortus GAPDH DNA vaccine with IL-12 adjuvant DNA vaccine Research Intramuscular injection (i.m.) In response to in-vitro stimulation by rGAPDH, splenocytes from mice vaccinated with rGAPDH or B. abortus S19 were able to produce gamma-interferon and tumour necrosis factor-a but not interleukin (IL)-4. Furthermore, the DNA vaccine partially protected mice against experimental infection.(Rosinha et al., 2002b) The B. abortus vaccine uses GAPDH with IL-12 adjuvant.(Rosinha et al., 2002b) Intramuscular injection (i.m.) B. abortus Omp16+IFA vaccines Subunit vaccine Research Intramuscular injection (i.m.) The vaccine uses B. abortus Omp19 and IFA adjuvant (Pasquevich et al., 2009). Intramuscular injection (i.m.) Recombinant protein preparation B. abortus Omp19 + IFA vaccine Subunit vaccine Research Intramuscular injection (i.m.) The B. abortus vaccine uses Omp19 and IFA adjuvant (Pasquevich et al., 2009). Intramuscular injection (i.m.) Recombinant protein preparation B. abortus P39 deletion vaccine VO_0000826 Live, attenuated vaccine Research Intramuscular injection (i.m.) A B. abortus vaccine that is recombinant B. abortus strain 19vaccine with the p39 gene deletion (Tibor et al., 1998). Intramuscular injection (i.m.) B. abortus pcDNA-BLS VO_0000421 DNA vaccine pcDNA3 [Ref416:Velikovsky et al., 2002] This is about pcDNA-BLS, a B. abortus DNA vaccine encoding lumazine synthase (Velikovsky et al., 2002). No. The Brucella abortus lumazine synthase (BLS) gene was cloned into the pcDNA3 plasmid. It is driven by the cytomegalovirus promoter (Velikovsky et al., 2002). Brucella abortus lumazine synthase (BLS) (Velikovsky et al., 2002). It was was cloned and identified in 1995 (Hemmen et al., 1995). DNA vaccine preparation The Brucella abortus lumazine synthase (BLS) gene was cloned into the pcDNA3 plasmid. This gene is driven by the cytomegalovirus promoter. The plasmid was amplified in E. coli JM109 (Promega, Madison, Wis.) and isolated with Mega Prep plasmid isolation columns (Qiagen, Dorking, United Kingdom) (Velikovsky et al., 2002). VO_0000286 Two independent protection experiments demonstrated that mice given pcDNA-BLS had a significantly higher degree of protection (1.65 log increase in protection) than did controls receiving PBS (P < 0.01) (Velikovsky et al., 2002). B. abortus strain 19 VO_0000022 Attenuated live vaccine Licensed USA; European countries Strain 19 induces reasonable protection against B. abortus, but at the expense of persistent serological responses (Schurig et al., 2002). Although effective, strain 19 vaccine has a tropism for the placenta and causes abortion when given to pregnant cows , is infectious for humans , and causes serologic responses in calves that are the same as those in cattle infected with natural field strains (Cheville, 2000). Strain 19, first described in 1930, is a laboratory-derived strain attenuated by an unknown process. It was originally isolated from bovine milk as a virulent strain in 1923, but became attenuated after storage at room temperature for over a year. Strain 19 is able to induce protective immunity in cattle (Schurig et al., 2002). Whole Brucella organism Mature female cattle were immunized with strain 19 ( 2x10^8 and 6x10^8 CFU respectively) and 6-12 month old calves with strain 19 ( 4x10^10 and 12x10^10 CFU ) . Mature females and calves were inoculated with sterile vaccine diluent and formed a non-vaccinated, in-contact control group (Geong et al., 2000). Of the 599 animals bled initially, 83.5, 72.6, 73.8 and 64.1%, respectively, were represented at 3, 6, 9 and 24 months post infection, respectively. No significant differences between the two villages in the percentage of animals presented at each sampling were found. During the trials, no difference was observed in the presenting of animals identified as seropositive and those identified as seronegative (Geong et al., 2000). Cattle were allowed to graze for 24 months under conditions where infection was possible (Geong et al., 2000). Mice (10/treatment group) were injected i.p. with 0.2 ml of a 0.15 M NaCl saline solution (controls) or with 0.2 ml of saline containing approximately 1x10^5 cfu of strain 2308 or 1x10^7 cfu of strain 19 or RB51. Blood samples and spleens were taken from age-matched noninfected control mice and from infected mice at 2, 4, 6, 10, or 20 weeks respectively after infection (Stevens et al., 1994b). No challenge performed. B. abortus strain 45/20 VO_0000723 attenuated live vaccine B. abortus strain 45/20 is a rough form of the bacterium that can confer immunity (Moriyon et al., 2004). The original strain of 45/20 was known to revert to smooth pathogenic forms when injected into cattle. Due to this instability, the vaccine is not currently marketed (Moriyon et al., 2004). The 45/20 strain was isolated from guinea pigs in 1938 (Moriyon et al., 2004). Whole Brucella organism 15 lambs were vaccinated with 45/20 bacterin, and a control group remained unvaccinated. Revaccinations were performed 18 weeks later. At regular intervals after vaccination and revaccination, blood samples were taken and animals were examined for genital lesions (Blasco et al., 1993). Two weeks after challenge, ten of the 15 control rams excreted B. ovis. All animals were slaughtered 15 weeks after the challenge inoculation and subjected to bacteriological and histological examinations. A ram was classified as protected if no B. ovis was isolated from any of the 13 organs sampled at necropsy. Significant levels of protection in comparison with the control group were obtained by vaccination with 45/20 bacterin (PC 0.05) (Blasco et al., 1993). One ram died from enterotoxemia during the course of the experiment (Blasco et al., 1993). Challenge inoculations were performed 31 weeks after the second vaccination with a virulent strain of B. ovis obtained from a naturally infected ram. Each ram received a total volume of 60 uL ( 1 x 10^9 CFU), as shown by viable cell counts made on the day of inoculation), both conjunctivally (30 uL) and preputially (30 uL) (Blasco et al., 1993). B. abortus subunit vaccine using L7/L12 VO_0000323 Subunit vaccine Maltose binding protein (MBP) (Oliveira et al., 1996). None The recombinant Brucella abortus L7/L12 ribosomal protein was fused to maltose binding protein (MBP). The animals vaccinated with MBP-L7/L12 received 25 mg of the specific B. abortus L7/L12 ribosomal protein. Different protein concentrations per dose containing the same MBP amounts were used to vaccinate each group to ensure that both mouse groups received the same amount of MBP (Oliveira et al., 1996). B. abortus ribosomal protein L7/L12 (Oliveira et al., 1996). Recombinant protein preparation Recombinant Brucella abortus L7/Ll2 ribosomal protein was fused to maltose binding protein (MBP). The detail is described (Oliveira et al., 1996). There was a specific antibody response from animals immunized with recombinant MBP-L7/L12 fusion protein. Serum samples from mice immunized with MBP-L7/L12 contained anti-MBP and anti-L7/L12 antibodies. No anti-MBP-L7/L12 antibodies were detected in the sera of naive mice used as a negative control (Oliveira et al., 1996). BALB/c Five groups of 15 BALB/c mice were immunized intraperitoeally with 0.2 ml of the following treatments: either phosphate-buffered saline (PBS); Immuneplus adjuvant system; adjuvant containing 75 micrograms of maltose binding protein (MBP); 4 adjuvant containing 100 micrograms of MBP-L7/L12 fusion protein; or B. abortus strain 19 (Oliveira et al., 1996). Three immunizations with 100 micrograms of the recombinant L7/L12 ribosomal protein fused to MBP protected mice against B. abortus infection, while vaccination with 75 micrograms of MBP alone resulted in no protection. The recombinant L7/L12 protein engendered substantial protective immunity to BALB/c mice against challenge when compared to the protection levels conferred to mice vaccinated with B. abortus S19. The greatest levels of protection observed in immunized mice were at 2 and 4 weeks post-challenge (Oliveira et al., 1996). The irrelevant Escherichia coli protein (MBP) does not protect mice against brucellosis (Oliveira et al., 1996). Mice were challenged one week after their final immunization (day 28) with an i.v. injection of 1 x 10^6 c.f.u. B. abortus in 100 microliters of sterile phosphate buffer saline (Oliveira et al., 1996). Activation of Th1 cell response. (Abtahi et al., 2008) BALB/C Three intra-muscular vaccinations of DNA vaccine encoding L7/L12 protein at 3 week intervals.(Abtahi et al., 2008) Lower bacterial cfu from vaccinated mice in comparison with control groups show the efficiency of L7/L12 DNA vaccination in mice model. (Abtahi et al., 2008) Splenic lymphocytes from L7/L12pCDNA3-vaccinated mice produced high levels of IFNy (3100 pg mL(-1)) 3 weeks post-vaccination. (Abtahi et al., 2008) Splenic lymphocytes from L7/L12pCDNA3-vaccinated mice produced low levels of IL-5 (300 pg mL(-1)) 3 weeks post-vaccination. (Abtahi et al., 2008) L7/L12pCDNA3 immunizations elicited high IgG2a isotype response in mice immunized (Abtahi et al., 2008) IgG1 titers induced post-immunization with L7/L12pCDNA3 vaccine. (Abtahi et al., 2008) B. abortus vaccine strain RB51 RB51 Colorado Serum Company CZ Veterinaria VO_0000021 Live attenuated B. abortus strain Licensed USA, Mexico, South American, India, Spain, Middle East, Iran RB51 has been the official cattle Brucella vaccine in the USA since 1996 and now in many other countries (Schurig et al., 2002). RB51 is a rifampin-resistant rough attenuated mutant of Brucella abortus derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. Rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop specific antibodies for the Brucella O-chain (Schurig et al., 1991). RB51 is a rough attenuated strain (Schurig et al., 1991). The antigen for this live attenuated vaccine is the whole cells of strain RB51. This strain has rough characteristics and is devoid of O-chain. The RB51 was derived from the virulent smooth B. abortus 2308 by several passages in media supplemented with sub-inhibitory concentrations of rifampicin (Schurig et al., 1991). BALB/c mice ~4 x 10^8 CFU Inoculation (ip) of RB51 into mice results in a splenic colonization which is cleared within four weeks post infection. RB51 does not revert to smooth colony morphology upon passage in vivo (mice) or in vitro (Schurig et al., 1991). The CFU in spleen decreases 1-2 logs. No obvious symptoms were observed in immunized mice. Vaccination of mice with RB51 induces specific cytotoxic T lymphocytes (CTLs) against strain RB51 and strain 2308-infected J774.A1 macrophages but not against Listeria monocytogenes-infected J774.A1 cells. Antigen-specific cytotoxic activity is exerted by T lymphocytes but not by NK cells. CD3+ CD4+ T cells secrete the highest level of IFN-γ and induce low but significant level of lysis of Brucella-infected macrophages. In contrast, CD3+ CD8+ T cells secrete low levels of IFN-γ but high levels of specific lysis of Brucella-infected macrophages. No nonspecific lysis was observed. Hence CD3+ CD4+ and CD3+ CD8+ T cells play synergistic roles in anti-Brucella activity (He et al., 2001). IFN-gamma is up-regulated in RB51-immunized mice (He et al., 2001). Sera from all animals of RB51 vaccinated and control groups did not show anti-Brucella antibodies on Days −30, 0, 15, and monthly thereafter until the day of challenge (Poester et al., 2006). Crossbreed At Day 0 of the experiment, heifers in the vaccinated group were divided in two sub-groups: 12 heifers were vaccinated at Day 0 of the experiment and the remaining 8 heifers were vaccinated at the 60th day of gestation, with a 2 mL dose. The heifers of the control group received 2 mL of sterile saline solution (Poester et al., 2006). Vaccination with RB51 vaccine did not result in the production of any antibodies against the O-side chain of lipopolysaccharide. The results indicate that vaccination with RB51 prevented 59.4% of abortions, 58.6% of cow infections, and 61.0% of fetal infections. The relative risk revealed that non-vaccinated animals have 2.462 times higher risk of aborting than RB51-vaccinated animals (Poester et al., 2006). All animals were challenged with the virulent B. abortus strain 2308. Each heifer received a 3.0 × 10^7 CFU challenge per heifer (Poester et al., 2006). During the study period, supramammary (N = 303) and retro-pharyngeal (N = 342) lymph nodes and spleen (N = 298) samples from 343 sero-positive animals slaughtered due to brucellosis suspicion were submitted for culture. Brucella was isolated from 176 animals (51%). Similar isolation rates (39%) were observed for supramammary and retro-pharyngeal lymph nodes, but a lower rate was seen for spleen (7%) (Martins et al., 2009). Mass vaccination of the cattle population of 3 Azore islands (Terceira, S. Miguel and S. Jorge) targeted breeding herds with the RB51 vaccine supplied by CZ Veterinaria S.A (Pontevedra, Spain). It was administered to heifers or adult female cattle s.c. at 10–34 × 10^9/dose (2 ml). In herds still infected with an intra-herd incidence of more than 10% after 6 months, all adults were re-vaccinated, as were adjacent herds (Martins et al., 2009). During the study period, supramammary (N = 303) and retro-pharyngeal (N = 342) lymph nodes and spleen (N = 298) samples from 343 sero-positive animals slaughtered due to brucellosis suspicion were submitted for culture. Brucella was isolated from 176 animals (51%). Similar isolation rates (39%) were observed for supramammary and retro-pharyngeal lymph nodes, but a lower rate was seen for spleen (7%) (Martins et al., 2009). The overall trend is a regular decrease of the three parameters from the beginning of the vaccination programme up to the end of the study period. Average herd incidence, herd prevalence and animal prevalence decreased 69.26%, 39.26% and 75.41% respectively, from 2002 to 2007 for the whole area. Mean within-herd prevalence followed the same pattern, declining on a gradual pace until 2007 (Martins et al., 2009). No side-effects, such as abortion were reported (passive reporting). The only outstanding observation that could be related to vaccination was a weak newborn calf from which RB51 was isolated. The annual number of human cases was also low (≤4) and none of them was due to RB51, but to field strain B. abortus biovar 1. It was impossible to relate the disease occurrence in humans with the prevalence in animals (Martins et al., 2009). n/a (Martins et al., 2009) RB51 vaccine supplied by CZ Veterinaria S.A (Pontevedra, Spain) (Martins et al., 2009). To establish the efficacy of RB51 in cattle, female calves were inoculated SC at 3, 5, 7, and 10 months, respectively with RB51 (n = 26), S19 (n = 22), or saline (n = 15) (Cheville, 2000). B. abortus strain RB51 was cultured from biopsies of superficial cervical lymph nodes in heifers vaccinated with RB51 and S19 at 10 weeks, but not at 12 weeks (Cheville, 2000). After vaccination, heifers administered RB51 developed no serum antibodies that reacted in the agglutinate test , but yielded a positive dot -blot assay using RB51 antigen . B. abortus strain RB51 was cultured from biopsies of superficial cervical lymph nodes from cows challenged with RB51and S19 vaccines at 10 weeks , but not at 12 weeks. Three month old vaccinated heifers were protected from infection and abortion; strain 19-vaccinated heifers were not infected nor aborted. Control heifers were infected. Vaccination at five and seven months of age gave equivalent, but less than complete protection. Heifers given strain 19 (n = 16) were protected 95%; those given RB51 were 87% protected; controls (administered saline) (n = 15) exhibited a high incidence of infection and abortion. No significant differences in the efficacy of either vaccine was found in animals at any given age. However, when compared to controls marked differences in the efficacy of each vaccine were noted. No gross or microscopic evidence of brucellosis was found in the tissues and organs of cows not infected at birth (Cheville, 2000). All heifers vaccinated with RB51 at three months were protected against infection and abortion. Vaccination at five and seven months of age yielded equivalent protection. Heifers given strain 19 were 95% protected. In calves, the results obtained suggest that RB51 protects at dosage levels comparable to those of strain 19.(Cheville, 2000). In cattle, RB51 is less virulent than B. abortus cattle vaccine Strain 19. Calves were bred at 16 to 17 months of age and challenged during the first pregnancy with virulent B. abortus (Cheville, 2000). Strain RB51 is protective in cattle at doses comparable to those of strain 19 (Cheville, 2000). Brucella abortus strain RB51 is the vaccine of choice against brucellosis of cattle in the United States . Thirty Brucella-free female buffaloes were divided in three groups. Group A consisted of 10 non-pregnant water buffaloes 4–5 years old that were vaccinated once with RB51 (10.2 × 10^10 CFU/ml) during lactation. Group B consisted of 10 adult water buffaloes 28–36 months old at the first lactation that were previously vaccinated twice (4 weeks apart) as calves (6–9 months old) with 10.2 × 10^10 CFU/ml RB51, but were not revaccinated with RB51 as adults. Group C consisted of 5 adults 4–5 years old during the fourth month of lactation and 5 adult water buffaloes 28–36 months old at the first lactation that were not vaccinated (controls) (Longo et al., 2009). RB51 was identified from milk samples collected during the first week post-vaccination (days 1 and 4) in 3/10 of the vaccinated animals of group A but was never isolated in groups B and C. Moreover, RB51 DNA was detected during the first week until the fourth week post-vaccination in the milk samples from group A but was not detected in milk samples of the animals of B and C (Longo et al., 2009). No side effects attributable to vaccine administration were noted in any treatment group. All the experimental water buffaloes appeared clinically normal throughout the study (Longo et al., 2009). On examination, all livers displayed hepatocellular vacuolization with a pronounced centrilobular pattern consistent with lipidosis and occasional multifocal areas of pleocellular infiltrates consisting of small and large mononuclear cells, suggesting mild hepatitis. One animal in the RB51 group also had focal necrotizing hepatitis. Other lesions observed in these animals included multifocal interstitial pyogranulomatous nephritis and nephrosis, focal necrotizing pancreatitis, and sparse mononuclear cellular infiltrates in the oviduct and uterus (Nol et al., 2009). Richardson's ground squirrel Each Richardson’s ground squirrel received 250 µl of inoculum directly into the mouth via tuberculin syringe. All squirrels were observed twice daily for clinical signs for 21 days and once daily thereafter. RB51, strain 19 (S19), and virulent B. abortus strain 9941 (S9941) were administered orally to squirrels to further characterize B. abortus infection in this species. Six groups of nongravid ground squirrels were orally inoculated with 6 x 10^8 CFU RB51, 2.5 x 10^4 CFU S19, 2.5 x 10^7 CFU S19, 1.3 x 10^6 CFU S9941, 2.1 x 10^8 CFU S9941, or vaccine diluent (control) (Nol et al., 2009). One of 5 animals in the lower-dose S19 group and 2 of 3 animals in the higher-dose S19 group showed persistence of bacteria in various tissues at 14 weeks post-inoculation (WPI). At 18 WPI, 1/5 animals in the RB51 group and 1/5 animals in the high-dose S9941 group were culture positive. There was a general lack of persistence of the three strains of B. abortus in Richardson’s ground squirrels at 18 wk PI (Nol et al., 2009). It is unknown if and when the three animals that were culture positive at 14 wk and the two positive animals at 18 wk may have cleared the infection if given more time. No evidence of shedding of RB51 or S19 beyond 2 days PI, and only 2/25 animals cultured at 18 wk showed evidence of persistent infection. Even if infected, squirrels are unlikely to be severely affected by B. abortus or serve as sources of Brucella in the field (Nol et al., 2009). There was no evidence of pathology caused by B. abortus strains in nonpregnant squirrels based on clinical signs, gross lesions, and microscopic lesions (Nol et al., 2009). Richardson’s ground squirrels experience minimal clinical signs and pathology at challenge levels ranging from 2.5 x 10^4 CFU to 6 x 10^8 CFU (Nol et al., 2009). In summary, RB51, S19, and S9941 do not produce disease in nonpregnant Richardson’s ground squirrels when given orally at challenge doses ranging from 2.5 x 104 cfu to 6 x 108 cfu. This species is unlikely to act as a reservoir or source of infection for either vaccine or wild type strains of B. abortus. The Richardson’s ground squirrel would be a poor model for oral Brucella infection as well. These data provide useful information for researchers and those charged with the management of brucellosis in wildlife in the GYA (Nol et al., 2009). Humoral responses post-vaccination (immunoglobulin G [IgG] levels), assessed at different time points, indicated that capsules containing live RB51 elicited an anti-Brucella specific IgG response. Furthermore, the encapsulated vaccine elicited a cell-mediated (CMI) response that the non-encapsulated vaccinates failed to produce (Arenas-Gamboa et al., 2009). Red deer (Cervus elaphus) A novel approach of immunization was examined using alginate composite microspheres containing a non-immunogenic, eggshell-precursor protein of Fasciola hepatica (Vitelline protein B, VpB) to deliver live RB51. 54 1–2-yr-old female red deer (Cervus elaphus elaphus), used as a model for elk, were vaccinated orally (PO) or subcutaneously (SC) with 1.5 x 10^10 viable organisms per animal. Specifically, animals were randomly distributed into six different treatments (n=9/group). Three groups were inoculated SC with a total dose of 1.5 x 10^10 CFU of either nonencapsulated RB51, encapsulated RB51 with alginate, or encapsulated RB51 with alginate and VpB. Two groups were vaccinated PO; one group received 1.5 x 10^10 CFU of encapsulated RB51 with alginate, and the second group received with encapsulated RB51 with alginate and VpB. The control group received a SC injection of 1 ml of empty capsules (no bacteria entrapped). A single vaccination dose was given to all animals (Arenas-Gamboa et al., 2009). At 12 wk postvaccination, animals that received the encapsulated vaccine with VpB in the formulation (regardless of the immunization route) were the only individuals that had a statistically significant proliferative response compared with the controls (P<0.0005 PO vaccinates, P<0.005 SC group; Fig. 2). Interestingly, the cpm counts in animals that received encapsulated RB51 with VpB PO were also higher than in deer that received the same formulation via SC (P<0.3). None of the animals that received nonencapsulated vaccine had a significant cellular response compared with naïve nonvaccinated animals. Immunization with RB51 elicited an anti-Brucella IgG response that was clearly detectable by 6 wk postvaccination. During the initial 17 wk, anti-Brucella IgG levels were higher in animals that received SC vaccine compared with the groups that were immunized PO. Between 17 to 28 wk, anti-Brucella IgG levels in animals that were PO-vaccinated had an increase in anti-Brucella IgG compared with deer SC-vaccinated (Arenas-Gamboa et al., 2009). Only animals that received encapsulated RB51 vaccine (either route) exhibited significant reduction in bacterial counts in their spleens. These data suggest that alginate-VpB microspheres provide a method to enhance the RB51 vaccine performance in elk. At 2 wk postchallenge, only animals that received encapsulated SRB51 with VpB had a significant decrease in bacterial load in the spleen. Red deer that received the vaccine PO were the only group that was statistically significant compared with the nonencapsulated, injected RB51. Animals that were PO-immunized with the VpB capsules had a 1.27 log reduction in spleen counts compared with animals vaccinated with nonencapsulated RB51 and a 1.68 log reduction compared with naïve, nonvaccinated, but S19 exposed, animals. S19 spleen counts in deer that received the VpB capsules via SC were also diminished by 1.21 log compared with the nonencapsulated RB51 and by 1.62 log compared with non-RB51 vaccinated controls (Arenas-Gamboa et al., 2009). At 7 mo postvaccination, three to four red deer from each vaccination group (except RB51/alginate SC; n=2) were challenged with a dose of 1x10^9 B. abortus strain 19 organisms by conjunctival exposure. Dose exposure was confirmed by serial dilutions and plating onto TSA plates. At 2 wk post-challenge, animals were euthanized, and spleens were harvested, weighed and homogenized. At 3–5 days postincubation, bacteria were enumerated (Arenas-Gamboa et al., 2009). Bison (Bison bison) CD1 Four groups of 10 female CD1 mice per group (5-6 weeks old) were vaccinated intraperitoneally (IP) with 3-5 x 10^8 CFU in 100 µl of RB51, RB51leuB, RB51leuB/pNS4, or RB51leuB/pNS4/GFP. Another group of 10 mice were vaccinated with saline to serve as a negative control. Three mice from each group were bled at 5 weeks post-vaccination to harvest serum. The sera were screened for GFP-specific antibodies by immunoblot (Rajasekaran et al., 2008). Mice vaccinated with the leuB mutant and the complemented RB51leuB strains were able to clear the virulent challenge strain B. abortus 2308 (S2308) at significant rates compared with clearance in the mice vaccinated with saline. No significant difference was observed in the rates of clearance of the challenge strain in the mice vaccinated with auxotrophic vaccine strains and the standard strain RB51 and also between the RB51leuB strains. In a separate experiment, the leuB auxotroph and the complemented leuB auxotroph were found to be cleared from CD1 mouse spleens by 4 to 5 weeks, at the same rate as the parent vaccine strain RB51 (Rajasekaran et al., 2008). The leuB/GFP construction was shown to complement a leucine auxotroph of RB51, which protected CD1 mice from virulent B. abortus 2308 (S2308) and elicited GFP antibodies. The leuB auxotroph and the complemented auxotroph of strain RB51 were able to protect the CD1 mice against an S2308 challenge. There was no significant difference in the protection levels (as measured by splenic clearance) afforded by the leuB auxotroph when the protection levels in the mice vaccinated with the complemented leuB auxotroph and with the complemented leuB auxotroph expressing GFP were compared to the protection level in the mice vaccinated with strain RB51. There was, however, a significant difference in protection afforded between the mice vaccinated with any of the RB51 strains and the saline control. Only sera from the group inoculated with RB51leuB/pNS4/GFP possessed GFP-specific antibodies (Rajasekaran et al., 2008). At 6 weeks post-vaccination, all groups of mice were challenged IP with 4 x 10^4 CFU of B. abortus strain 2308 (S2308). At 2 weeks post-challenge, mice were euthanized by CO2 asphyxiation, and their spleens were recovered, homogenized, serially diluted, and plated on trypticase soy agar (TSA) plates to estimate CFU (Rajasekaran et al., 2008). (Olsen et al., 2007) BALB/c To characterize the optimal aerosol dosage of Brucella abortus strain 2308 (S2308) and B. melitensis (S16M), 10^3–10^10 CFU were nebulized to BALB/c mice. A range of dosages was evaluated to determine optimal dosages for aerosolization. Protection in vaccinated and non-vaccinated mice were compared after experimental B. abortus challenge via IP and aerosol routes. For aerosol delivery, the nebulization apparatus consists of a compressed air tank and a jet nebulizer connected via a rubber hose to a port in a metal cage cover. A flow rate of 2 l/min produces a 5 ml particle size. Compressed air was used to jet nebulize 2 ml of challenge inoculum directly into a plexiglass cage containing 5 mice. Nebulization continued until all inoculum, plus 2 ml saline was delivered over ~15 min. Experiment 1: S2308 or S16M were spectrophotometrically adjusted to concentrations between 10^3 and 10^10 CFU and delivered by aerosol to female 10-week-old BALB/c mice. At 2 weeks after nebulization, mice were euthanized with CO2. Spleen, liver, and lung from each mouse were removed and weighed. Tissues were macerated and serially diluted, with standard plate counts used to determine bacterial concentrations. Experiment 2: Mice were injected IP with 0.2 ml of saline +/- ~1 x 10^7 CFU of SRB51. At 12 weeks after vaccination, mice were challenged with S2308 IP (0.2 ml saline + 5 x 10^4 CFU) or via nebulization (~10^9 CFU). Two weeks after challenge, mice were euthanized with CO2. Spleens, livers and lungs were weighed and processed for isolation and quantification of Brucella as described in Experiment 1. (Olsen et al., 2007) Total CFU per tissue increased beginning at 10^6–10^7 CFU dosages, with 10^9 CFU appearing to be an optimal dosage for S16M or S2308 aerosol delivery. (Olsen et al., 2007) Mice vaccinated with SRB51 had reduced splenic, liver and lung colonization after IP challenge with S2308 as compared with control mice after IP S2308 challenge. Control and SRB51-vaccinated mice did not differ in splenic, liver, or lung colonization after aerosol S2308 challenge. (Olsen et al., 2007) (Olsen et al., 2007) At 12 weeks after vaccination with 10^7 CFU of SRB51 or saline (control), mice were challenged IP with 6.4 x 10^4 CFU or via aerosol (1.76 x 10^9 CFU) with S2308. (Olsen et al., 2007) (Olsen et al., 2007) (Pasquali et al., 2003) BALB/c Investigators evaluated the effect of oral inoculation (PO) of Brucella abortus RB51 in BALB/c female mice against a challenge infection with B. abortus (S)2308. For IP immunization with live bacteria, mice were given 2 x 10^8 CFU of B. abortus RB51 in 0.2 ml of sterile saline or 2 x 10^4 CFU of strain S2308. For PO immunization, a gastric lavage needle was used. Ten minutes prior to oral inoculation, mice were administered 0.2 ml of 10% sodium bicarbonate (NaHCO3) to neutralize gastric acidity or 0.2 ml of sterile saline. Mice were inoculated PO with 2 x 10^10 CFU of RB51 or S2308. On days 10 and 18 after PO RB51 or S2308, preceded or not by gastric acidity neutralization, mice were killed and their spleens removed. Spleens were homogenized in 1 ml of sterile saline. Aliquots of the resulting suspensions were plated to assess spleen colonization. In separate experiments, mice were inoculated PO with RB51. Oral inoculation was preceded by gastric acidity neutralization. To evaluate the excretion of bacteria after oral exposure, fecal samples were collected daily for 3 days after inoculation. At 7, 15, 30, and 42 days after infection, mice were bled and killed and spleens were aseptically removed, weighed, homogenized in PBS, and plated to determine the outcome of infection (Pasquali et al., 2003). Gastric acid neutralization prior to the oral inoculation contributed to a more homogeneous and consistent infection with both RB51 and S2308. Clearance and immune response following oral infection with RB51 was successively assessed. PO inoculation gave a mild infection, which was cleared 42 days later and induced a delayed humoral and cell-mediated immune (CMI) response (Pasquali et al., 2003). PO RB51 was able to protect mice infected with S2308 PO but not to mice infected IP. Results indicate that oral inoculation of mice with RB51 is able to give protective immunity against oral infection with virulent strains and that this protection seems to rely on an immune response at the mucosal level (Pasquali et al., 2003). (Pasquali et al., 2003) Finally, they immunized mice PO with RB51 and challenged them with S2308 PO or intraperitoneally (IP) 42 days after vaccination. Mice were PO vaccinated with RB51 preceded by gastric acidity neutralization or were IP inoculated with RB51 as mentioned above. Additional mice served as unvaccinated controls. PO and IP vaccinated mice and controls were challenged PO (2 x 10^10 CFU) or IP (2 x 10^4 CFU) with S2308 42 days after vaccination. PO challenge was preceded by gastric acid neutralization. Mice were bled and killed at 18 days after challenge. Spleens were weighed and homogenized to determine CFU as a means for assessing the protective response induced by vaccination. Sera were used to evaluate antibody titer against both S2308 and RB51 (Pasquali et al., 2003). (Pasquali et al., 2003) (Ko et al., 2002) C57BL/6 (IRF-1-/-) S2308 (the virulent wild-type Brucella abortus strain), S19 and RB51 (live attenuated vaccine strains), cyd and cbp (insertional mutants), and bap (deletion mutant) were used in this experiments. Interferon regulatory factor 1-deficient (IRF1-/-) mice were originally produced by using C57BL/6 (H-2b) mice. IRF1-/- mice were injected IP with diverse strains and CFU of B. abortus in 200 ul of PBS (Ko et al., 2002). To count residual CFU in the spleens or livers of mice, 5 mice from each group were examined at each sampling period. Brucella colonies were counted after a 3-day incubation. Serum glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were also quantitatively measured (Ko et al., 2002). Interferon regulatory factor 1-deficient (IRF1-/-) mice infected with virulent Brucella abortus (S)2308 at 5 x 10^5 CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF1-/- mice were unable to resolve infection and died. In contrast, IRF1-/- mice survived when infected at 5 x 10^5 CFU with several attenuated Brucella strains (S19, RB51, cbp, and cyd). Brucella-infected mice were also killed 12 days post-infection for histologic analysis (Ko et al., 2002). After 6 weeks, surviving IRF1-/- mice were challenged with 5 x 10^5 CFU of S2308 in 200 ul of PBS (Ko et al., 2002). (Ko et al., 2002) Antibody and cytokine responses were Th1-focused in young mice, but Th-mixed in old mice, including evidence of the Th17 subtype immune response (High et al., 2007). BALB/c, DBA/2 Young (2–3 month old) or old (16–18 month old) adult female mice (DBA/2 or BALB/c) were infected with either an attenuated Brucella abortus strain that over-expressed superoxide dismutase (RB51-SOD) or with a fully virulent wild-type strain (S2308). They were inoculated intra-peritoneally (IP) with 3.4 x 10^7 CFU or 6 x 10^8 CFU RB51-SOD, or with 2 x 10^4 or 4 x 10^9 CFU S2308. Uninfected control mice were injected with saline alone. Spleens harvested after sacrifice were cultured quantitatively for Brucella. Splenocytes (80–120 x 10^6) from mice were harvested and cultured in the presence of heat-killed B. abortus, purified SOD, or the mitogen concanavalin A (conA). Supernatants were assayed for the presence of cytokines by protein array and ELISA, semi-quantitatively measured by densitometry and the intensity of a given dot-blot compared to the mean of the positive control dots. Blood was obtained via retro-orbital bleeds. Total IgG, IgG1 and IgG2a titers vs. two Brucella proteins, bacterioferritin (BFR) and Cu++/Zn++ SOD, were assessed by ELISA (High et al., 2007). All young and old mice survived infection with RB51-SOD (up to 6 x 1^08 CFU) or S2308 (up to 8 x 10^8 CFU). Old mice had a lower organism burden in the spleen than young mice 5 or more weeks after infection (High et al., 2007). Immunization with the RB51-SOD strain provided protection vs. strain 2308 challenge in young and aged BALB/c, but only young DBA/2 mice (High et al., 2007). For protection studies, mice were either immunized with 4 x 10^8 CFU RB51-SOD or saline as a control and allowed to recover for 5 weeks. Mice were then infected with 2 x 10^4 CFU S2308 and sacrificed 2 weeks later for determination of CFU/spleen as above (High et al., 2007). BALB/c Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice vaccinated with RB51 gave negative results in the conventional serological tests prior to challenge, standard tube agglutination test (SAT), Rose Bengal plate test (RBPT), buffered acidified plate antigen test (BAPAT), and mercaptoethanol test (MET). Sero-conversion took place to a whole-cell bacterial buffered RB51 antigen after vaccination and persisted for 7 weeks post-vaccination. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Protection units were showed that Rev. 1 vaccine was superior to RB51 vaccine in protection of mice against B. melitensis (Hamdy et al., 2002). Mice were challenged with a B. melitensis field strain. The humoral immune responses induced in mice after vaccination and challenge were detected by the standard tube agglutination test (SAT), mercaptoethanol test (MET), Rose Bengal plate test (RBPT), and buffered acidified plate antigen test (BAPAT). The detection of immune responses induced by the RB51 vaccine was measured by RB51 killed antigen. Mice were bled under anesthesia from the retroorbital sinus or from heart puncture. Sera were stored at -70C unless used on the same day. Five animals from each of the first and the second groups were sacrificed by CO2 asphyxiation at weekly intervals. Detection of Brucella vaccinal strains in internal organs (spleen, liver and lung) was done at weekly intervals. Two weeks after elimination of the vaccinal strains from the internal organs and disappearance of any serological reactions, the remaining animals and control were challenged with 5 x 10^5 cfu of B. melitensis i.p. in 0.2 ml PBS. Five of the challenged animals from each group were sacrificed every week until the end of the experiment. Sacrificed animals were subjected to serological and bacteriological investigations. Bacteriological examinations and viable counts of the Brucella organisms per spleen following challenge were carried out. Identification of the rough form of the RB51 vaccinal strain was achieved by auto-agglutination reaction with acriflavine solution (1/1000) and the ability to stain with crystal violet (Hamdy et al., 2002). BALB/c This study determined if murine interleukin-12 (IL-12) would influence immunity in mice vaccinated with live or killed Brucella abortus strain RB51 (SRB51). Mice received live or irradiated SRB51 bacteria alone, or with IL-12 (0.5 or 1.0 jig, 2x or 3X), whereas other mice received saline or IL-12 alone. Specifically, female 10-week-old BALB/c AnNHsD mice (n = 12/group) were vaccinated intraperitoneally (IP) with 0.2 mL of saline containing 3 x 10^8 CFU of live SRB51 alone, or combined with 0.5 or 1.0 ug of murine recombinant IL-12, administered thrice (days 0, 5, and 21). Additional mice were vaccinated IP with 0.2 mL of saline containing 3 x 10^8 CFU of irradiated SRB51 alone, or combined with 0.5 or 1.0 ug of IL-12 administered twice (days 0 and 5) or thrice. Other mice were injected IP with 0.2 mL of saline alone or with saline containing 0.5 or 1.0 ug of IL-12 administered twice or thrice (Lee et al., 2001). Post-vaccination antibody responses to live or killed SRB51 and clearance of live SRB51 from splenic tissue were not influenced by IL-12 treatments (Lee et al., 2001). The highest IL-12 treatment increased post-challenge antibody responses when co-administered with killed SRB51. Co-administration of 1.0 ug of IL-12 with live SRB51, but not killed SRB51, reduced S2308 colonization of splenic tissues. Following euthanasia with C02/02 at 2 wk after S2308 challenge, blood, spleens, and livers were obtained from all mice. Spleens were weighed, approximately onehalf was excised for bacteriologic examination and weighed, and remaining tissue was used to prepare spleen cell suspensions. Following weighing, the entire liver was designated for bacteriologic examination. Tissue for bacteriologic examination was stored at -69°C until processed (Lee et al., 2001). Mice were challenged at 12 weeks with 4 x 10^4 CFU of B. abortus strain 2308 (S2308) and were euthanized 2 weeks later. Remaining mice (n = 8/trt) were challenged IP with 4 X 104 cfu of S2308 at 12 wk after vaccination. The time of challenge was based on data from previous studies which demonstrated that a dosage of 5 X 108 cfu of SRB51 is cleared from BALB/c mice by 12 wk after IP vaccination (Lee et al., 2001). After vaccination, both Th1 and Th2 cytokine patterns were observed. Of those, the early production of gamma interferon seems to have the prominent role in inducing an immunologically based protection. Spleen weight in vaccinated and infected mice did not increase after challenge and were not significantly different from nonchallenged, vaccinated animals killed 42 days after vaccination. In contrast, spleen weights of unvaccinated and infected mice showed a significant enlargement as early as 6 days after infection. Vaccinated mice were almost refractory to a subsequent challenge infection with B. abortus 2308 cells, exhibiting significantly lower levels of infection compared to unvaccinated infected animals after infection. Vaccination alone induced a number of significant effects upon cytokine expression. Spleen cells from unvaccinated mice stimulated with heat-inactivated RB51 cells did not produce measurable cytokines throughout the course of the experiment. Similarly, spleen cells from vaccinated mice killed 6 and 18 days after vaccination did not produce measurable bio-active IL-12 (p70) when stimulated in vitro, but a low level of production was detectable in spleen cells from mice killed 42 days after vaccination (Pasquali et al., 2001). BALB/c The present study was performed to delineate cytokine induction after intraperitoneal (IP) administration of Brucella abortus strain RB51 and subsequent infection with virulent strain B. abortus S2308. Twelve- to 14-week-old BALB/c female mice were allocated to experimental groups consisting of five animals each. Mice were vaccinated IP with 0.2 ml of saline containing 2 x 10^8 CFU of RB51. Unvaccinated control animals remained untreated throughout the experiment (Pasquali et al., 2001). Vaccinated mice had higher spleen weights when compared to control animals after vaccination. Vaccination of mice with live RB51 resulted in a pattern of bacterial growth in which peak numbers were seen 18 days after vaccination, followed by a progressive decline. Bacteria were absent in the spleen at 42 days postvaccination. Counts on days 6 and 18 were significantly different (Pasquali et al., 2001). RB51 confers a solid immunity that protects mice against a challenge infection with virulent B. abortus 2308. After vaccination, both Th1 and Th2 cytokine patterns were observed. Protection may be more dependent upon the timing of the cytokine response rather than on the absolute level of cytokine expression. Challenge infection of vaccinated and nonvaccinated mice induced even more marked increases in cytokine expression patterns. IL-12 p40 was detectable in spleen cells from both vaccinated and unvaccinated mice after challenge infection, and there were no significant differences between vaccinated and unvaccinated mice. Similar results were seen throughout the course of the experiment for IL-12 p70 production, which was low at all times in all groups of mice. In contrast, IFN- production was detectable as early as 3 days after challenge infection in spleen cells from both vaccinated and unvaccinated mice, although levels were significantly higher in vaccinated mice. Levels of IFN in spleen cells from vaccinated, challenged mice showed no differences throughout the experiment, in contrast to spleen cells from unvaccinated, challenged mice which showed increased IFN production that reached levels seen in spleen cells from vaccinated, challenged mice 6 days after challenge. After challenge infection, low levels of IL-4 were detectable, reaching a peak in spleen cells 6 days after challenge. However, levels were not different between vaccinated and unvaccinated mice. IL-10 showed a pattern similar to that of IFN-. IL-10 was detectable in both vaccinated and unvaccinated mice after challenge infection. However, vaccinated and challenged animals did not show any differences at different times after challenge. Conversely, IL-10 production in unvaccinated, challenged mice was first detectable 6 days after challenge infection and reached a peak 10 days after challenge infection. Results of this study indicated that mice vaccinated with RB51 cells were protected as early as 3 days after a challenge infection with virulent B. abortus 2308 cells. In vaccinated mice, challenge infection resulted in high levels of IL-10 production at all times tested. In contrast, in unvaccinated mice, IL-10 production reached levels comparable to that in the vaccinated mice beginning 6 days after infection. Previous studies indicate that both IL-10 and IFN- are produced following a B. abortus infection and that IL-10 induction does not down-regulate IFN- production. This implies that in brucellosis, the effect of IL-10 on the immune response is to limit the consequences of an exaggerated proinflammatory response more than to counterbalance the production of Th1 cytokines. These findings are in agreement with others which measured IL-10 production following Brucella infection (Pasquali et al., 2001). At 42 days after vaccination, unvaccinated controls and IP vaccinated mice were challenged IP with 0.2 ml of saline containing 2 x 10^4 CFU of B. abortus 2308. At 6, 18, and 42 days after vaccination and 3, 6, and 10 days after challenge, mice were euthanatized and spleens were aseptically removed. Approximately one-third of the spleen was weighed and homogenized in PBS, and an aliquot of the resulting cell suspension was plated to determine the number of CFU. The remaining 2/3 of the spleens were weighed, minced, and used to prepare spleen cell suspensions. Cytokine expression was assayed in culture supernatants of splenocytes stimulated with 10^8 heat-inactivated B. abortus RB51 or 2308 bacteria per well. Cytokines were detected by ELISA according to the manufacturer’s instructions (Pasquali et al., 2001). Nine of the 20 (45%) orally inoculated (PO) RB51 mice necropsied in the first 4 wk post-inoculation (WPI) had mild multifocal pleocelluar inflammatory infiltrates in the liver; 3/25 (12%) PO RB51 mice necropsied after 4 WPI had similar lesions. All 10 of the RB51 IP mice necropsied in the first 2 WPI had multifocal pleocellular inflammatory infiltrates in the liver; 2/10 (20%) RB51 IP mice necropsied 3-4 WPI had similar lesions, and only 1 necropsied after 4 WPI from this group had these lesions. Generally, lesions in the IP group were more severe (more cellular infiltrates) than in the PO RB51 group. Lesions were milder in mice at longer times PI. One PO RB51 male mouse necropsied 5 WPI had a mild lymphocytic infiltrate in the interstitium of the seminal vesicle, but no RB51 was recovered from the spleen. Culture results were usually correlated with histologic lesions. Mice with the highest number of colonies of RB51 isolated were those with the most severe lesions (Cook et al., 2001). Deer mouse (Peromyscus maniculatus) This study was designed to determine effects of strain RB51 on deer mice (Peromyscus maniculatus), a nontarget species that could have access to treated baits in a field situation. 90 mice were orally dosed (PO) or intraperitoneally injected (IP) with 1 x 10^8 colony forming units (CFU) strain RB51, and 77 controls were similarly dosed with sterile saline. At weekly intervals for 2 months, 4-6 mice from each group were euthanized, gross necropsies performed, spleens and uteruses cultured, and tissues examined histologically (Cook et al., 2001). All PO inoculated mice cleared the infection by 6 weeks post-inoculation (wpi). While most of the injected mice cleared the infection by 7 wpi, a few required 9 w. With one possible exception (see below), no lesions attributable to brucellosis were noted on gross necropsy. Mice orally inoculated with RB51 developed low level splenic infections with strain RB51, but all were negative by 6 wk PI. RB51 was isolated from the uterus of one orally inoculated mouse necropsied 2 wk PI; no other orally inoculated mouse had a uterine infection. IP inoculated mice had a higher rate of splenic and uterine infections and took longer to clear the infections than orally inoculated mice with some remaining positive at 8 wk PI (Cook et al., 2001). There were minimal adverse effects attributable to strain RB51. RB51 would not negatively impact P. maniculatus populations if it were used in a field situation. Incidental findings found in some mice in all groups included: mild myocarditis (19% of controls; 30% of RB51 inoculates), mild metritis (10% of controls; 5% of RB51 inoculates), mild multifocal pyogranulomatous pneumonia (8% of controls; 5% of RB51 inoculates), and nematodes in the small intestines and/or cecum (54% of controls; 33% of RB51 inoculates). In a field situation, deer mice would only be exposed to the vaccine PO and not IP. All the orally exposed animals remained clinically healthy with no evidence of illness. Orally exposed mice had fewer CFU cultured from their spleens and uteruses, cleared the vaccine more quickly, and had milder lesions than mice inoculated IP. Strain RB51, with the possible exception of one IP inoculated animal, did not cause significant morbidity or mortality (Cook et al., 2001). Deer mice (Peromyscus maniculatus) Deer mice (Peromyscus maniculatus, n = 14) were orally inoculated (PO) with Brucella abortus strain SRB51 or physiologic saline. Oral and rectal swabs and blood samples were collected for bacteriologic evaluation. Rodents were necropsied at 8-10 wk and 12-21 wk post inoculation (WPI). Spleen, liver and reproductive tissues were collected for bacteriologic and histopathologic evaluation (Januszewski et al., 2001). No differences in clinical signs, appetite, weight loss or gain, or activity were observed between saline- and SRB51-inoculated animals. Oral and rectal swabs were negative throughout the study. In tissues obtained from SRB51-inoculated animals, the organism was isolated from 1/6 (17%) deer mice necropsied at 8 WPI. SRB51 was not recovered from deer mice necropsied 12 WPI. SRB51 was not recovered from saline-inoculated deer mice at any time. Results indicate oral exposure to SRB51 does not produce morbibity or mortality in deer mice (Januszewski et al., 2001). One SRB51-inoculated mouse died immediately after inoculation, and one control mouse died at 4 days PI. Deaths were attributed to handling and probable aspiration of the inoculum. Results indicate oral exposure to SRB51 does not produce morbibity or mortality in deer mice (Januszewski et al., 2001). RB51 vaccination of mice induces specific CTLs and suggest that CD3+ CD4+ and CD3+ CD8+ T cells play a synergistic role in the anti-Brucella activity. Specifically, Brucella abortus strain RB51 vaccination of mice induced specific cytotoxic T lymphocytes (CTLs) against both RB51- and virulent strain 2308-infected J774.A1 macrophages but not against Listeria monocytogenes-infected J774.A1 cells. The antigen-specific cytotoxic activity was exerted by CTLs but not by NK cells. CD3+ CD4+ T cells secreted the highest level of gamma interferon (IFN) and were able to exert a low but significant level of specific lysis of Brucella-infected macrophages. They also exerted a low level of nonspecific lysis of noninfected macrophages. In contrast, CD3+ CD8+ T cells secreted low levels of IFN-g but demonstrated high levels of specific lysis of Brucella-infected macrophages with no nonspecific lysis (He et al., 2001). BALB/c Six-week-old BALB/c female mice were inoculated intraperitoneally (IP) with 4 x 10^8 CFU of Brucella abortus strains RB51 or S2308 or the same volume of saline (negative control). The mice were euthanatized at 6 weeks postimmunization (wpi) by CO2 inhalation. These mice served as splenocyte sources for CTL assays. Splenocytes from strain RB51-infected or saline-inoculated mice were resuspended in 2 ml of c-RPMI and passed through nylon wool columns to enrich for cytotoxic T lymphocytes (CTLs). After 5-day incubation, cells were collected, and live effector cells were obtained by removing dead cells by Histopaque centrifugation. CTLs were also collected and used for flow cytometric analysis, magnetic cell sorting, and colorimetric CTL assay (He et al., 2001). RB51 vaccination of mice resulted in the development of a strong cytotoxic T-cell (CTL) response. The specific anti-Brucella cytolytic activity was mainly exerted by CD3+ CD8+ T cells. CD3+ CD4+ T cells also developed after immunization, secreted high levels of IFN, and exhibited certain levels of specific and nonspecific lytic activity against Brucella-infected target cells. NK cells appeared not to contribute significantly to the observed Brucella-specific cytotoxic activity (He et al., 2001). BALB/c This study was designed to determine if a single 0.5 ug administration of recombinant murine interleukin-12 (IL-12) would influence immune responses of mice vaccinated with live or killed Brucella abortus strain RB51 (SRB51). A total of 180 (n = 30/treatment) female 10-week-old BALB/c AnNHsD mice were vaccinated intraperitoneally (IP) with 0.2 mL of saline containing 5 x 10^8 cfu of live or gamma-irradiated SRB51 bacteria alone, or in combination with 0.5 ug of IL-12. Control mice received saline +/- 0.5 ug of IL-12 (Lee et al., 1999). Mice vaccinated with live SRB51 or live SRB51+IL-12 had greater serum antibody titers against SRB51 at 2, 4, 8, and 12 wpv when compared to all other treatments. When combined with live or killed SRB51, IL-12 as an adjuvant did not influence serologic responses at 2, 4, 8, or 12 wpv. In the absence of IL-12 as an adjuvant, spleen cells obtained at 12 wk after IP vaccination with live SRB51 had greater stimulation indexes against irradiated S2308 when compared to responses of spleen cells from other treatments. Administration of IL-12 as an adjuvant with live or killed SRB51 did not enhance lymphocyte proliferative responses to S2308 (Lee et al., 1999). The clearance of SRB51 from spleens of mice vaccinated with live SRB51 alone, or in combination with IL-12, did not differ at 2, 4, 8, or 12 wk after vaccination. At 12 wk after vaccination, spleens of mice vaccinated with either live SRB51 or live SRB51+IL-12 were culture negative for SRB51. IL-12 did not influence the clearance of strain RB51 from splenic tissue. Spleen weights of mice vaccinated with live SRB51 or live SRB51+IL-12 were greater at 2, 4, 8, or 12 wpv when compared to saline controls, IL-12 controls, or mice receiving either vaccination treatment using killed SRB51. Addition of exogenous IL-12 to culture supernatants of spleen cells obtained at 4 and 8 wk, but not at 2 or 12 wpv, enhanced lymphocyte proliferative responses of all vaccine treatments. Maximal enhancement of lymphocyte response was associated with the addition of IL-12. Mice vaccinated with live SRB51 (5 X 108 cfu) had lower spleen weights, total spleen cfu, and spleen cfu/g following challenge at 12 wk after vaccination when compared to mice receiving saline control, IL-12 control, or killed SRB51 treatments. Administration of IL-12 as an adjuvant with live or killed SRB51 did not enhance resistance against experimental challenge with S2308 (Lee et al., 1999). Mice (n = 10/treatment) were challenged IP with 2 X 10^4 cfu of S2308 at 12 weeks post-vaccination (wpv). Blood samples and spleens were obtained from mice in all treatments after CO2/O2 euthanasia at 2, 4, 8, and 12 wpv (n = 5/treatment/time). Following C02/02 euthanasia at 2 weeks after S2308 challenge (wpc), blood and spleens were obtained from mice in all treatments (n = 10/treatment). Cells from 5 mice from each treatment were evaluated separately at 2, 4, 8, and 12 wpv. Following S2308 challenge, spleen cell suspensions from 10 mice from each treatment were combined into 5 separate suspensions (2 mice/suspension, 5 suspensions/treatment) for evaluation of lymphocyte proliferative responses. Cell proliferation results were expressed as stimulation indices (counts/min in presence of antigen divided by counts/min in medium only) (Lee et al., 1999). Oral SRB51 vaccination induced lower levels of antibodies to the surface antigens of intact SRB51 bacteria than did IP vaccination (Stevens et al., 1996). BALB/c Immune responses and resistance to infection with Brucella abortus 2308 (S2308) were measured in mice following oral (PO) or intraperitoneal (IP) vaccination with strain RB51 (SRB51). Female 10-week-old BALB/c AnNHsD mice were used in the following experiments. Lyophilized SRB51 reconstituted in a 0.15 M NaCl saline solution was used to vaccinate PO by placing 20 ml of saline containing 5 x 10^8 or 5 x 10^6 CFU onto the pharyngeal mucosa. Additional groups of mice were vaccinated IP with 0.2 ml of saline containing 5 x 10^8 or 5 x 10^6 CFU of SRB51. Nonvaccinated control mice were injected IP with 0.2 ml of saline alone (Stevens et al., 1996). Bacteria persisted in the parotid lymph node for 4 weeks following PO vaccination of mice with 5 x 10^8 or 5 X 10^6 CFU of SRB51. Bacteria did not appear in the spleen during 12 weeks after PO vaccination, whereas they did appear in the spleen for 8 weeks following IP vaccination of mice with SRB51. Increased resistance to S2308 infection occurred at 12-20 weeks in mice vaccinated IP with SRB51 but occurred at 12 weeks only in mice vaccinated PO with 5 x 10^8 CFU SRB51 (Stevens et al., 1996). Neither route of vaccination induced anamnestic antibody responses to the surface antigens of intact S2308 bacteria after challenge infection of the vaccinated mice with S2308. Mice vaccinated PO with SRB51 and challenged with S2308 at 12-20 weeks had lower and less persistent spleen cell proliferation and production of gamma interferon (IFN) in response to S2308 and certain immunodominant S2308 proteins (<18-32 kDa) than did mice vaccinated IP with SRB51. However, mice had similar spleen cell tumor necrosis factor alpha (TNF) production. These results indicate that PO vaccination of mice with SRB51 was effective in inducing protective immunity to S2308 infection, although the immunity was lower and less persistent than that induced by IP vaccination. The lower protective immunity induced by oral vaccination may have resulted from lower and less persistent cell-mediated immunity and IFN production in response to S2308 and S2308 proteins (Stevens et al., 1996). Nonvaccinated control mice and vaccinated mice at 12, 16, or 20 weeks after vaccination were challenged with S2308 by an IP injection of 2 x 10^4 CFU in 0.2 ml of 0.15 M NaCl saline solution (Stevens et al., 1996). Spleens from RB51-infected mice showed only mild to moderate accumulations of macrophages in marginal zone areas during the peak of RB51 infection (PID 7-10). Morphometric analysis of RB51-infected spleens showed a decrease in white pulp area, which coincided with peak bacterial numbers. However, this decrease was not significant. Spleens from mice infected with htrA showed moderate to marked accumulations of macrophages in marginal zone areas, which persisted through PID 60. Multifocal necrosis in lymphoid follicles as early as PID 4 was seen in both htrA and 2308 infection (Palmer et al., 1996). BALB/c BALB/c mice were inoculated intraperitoneally (IP) with suspensions of Brucella abortus strains 2308 or RB51 or an htrA mutant. Spleens were examined on post-inoculation day (PID) 2, 4, 7, 10, 15, 21, 30, and 60. 132 male BALB/c mice were infected with either one of the 3 B. abortus strains or with saline as a control (Palmer et al., 1996). Brucellae were cultured in high numbers from the spleens of mice infected with strains 2308 or htrA through PID 60; however, mice infected with RB51 cleared the infection between PID 30 and 60. Histopathologic changes in spleens from 2308-infected mice were characterized by marked accumulations of macrophages, which expanded marginal zones beginning as early as PID 7 and persisting through PID 60. Morphometric analysis showed a decrease in splenic white pulp in 2308-infected mice at PID 10, which correlated with the peak of bacterial infection. Although this decrease was significant when compared with values at previous and following time periods, it was not significantly different from white pulp values noted at PID 2 or 4 or the values for control spleens (Palmer et al., 1996). Morphometric analysis of htrA-infected spleens revealed no significant decrease in white pulp and no obvious correlation with bacterial numbers in the spleen. These results suggest that virulent B. abortus does not induce lymphoid depletion significantly below those values seen in noninfected mice; thus, the possible role of lymphoid depletion in the pathogenesis of brucellosis remains questionable (Palmer et al., 1996). B. melitensis 16M DNA vaccine expressing Omp31 VO_0000325 DNA vaccine pTargeT [Ref688:Gupta et al., 2007] The strain isolated from the goat was virulent, bu the plasmid created was quite attenuated (Gupta et al., 2007). B. melitensis 16M, the virulent strain, was isolated from the stomach content of the aborted fetus of the goat. B. melitensis DNA fragment encoding omp31 was cloned in pTargeT mammalian expression system vector. The plasmid was then purified and made into a vaccine consisting of 100 μg of pTargeTomp31 in 50 μl sterile saline (Gupta et al., 2007). The antigen for this vaccine were found in eukaryotic expression vectors called pTargeTomp31, which encoded the outer membrane protein (omp31) of B. melitensis 16M (Gupta et al., 2007). DNA vaccine construction The nucleotide sequence of the gene coding for the outer membrane protein omp31 reported for B. melitensis were synthesized by Qiagen. The primers were used to amplify a target sequence of 720 bp within a gene code for the production of a 28–31 kDa outer membrane protein (omp31) specific to the B. melitensis. The omp31 gene of B. melitensis was amplified with primers 5′-TGACAGACTTTTTCGCCGAA-3′ and 5′-TATGGATTGCAGCACCGC-3′. This 720 bp B. melitensis DNA fragment encoding omp31 was cloned in pTargeT mammalian expression system vector. The resultant plasmid (pTargeTomp31) contained the omp31 gene. Competent Escherichia coli JM109 was transformed with pTargeTomp31. Ampicillin-resistant colonies were grown in Luria-Bertani medium containing 100 μg/ml of ampicillin at 37 °C with agitation at 225 rpm. Protein expression was induced by adding 1 mM isopropyl-β-d-thiogalactopyranoside and incubating transformed cells for 4 h. Bacteria were pelleted by centrifugation and frozen at −20 °C. Bacterial cells were suspended in a solution consisting of 50 mM Tris, 5 mM EDTA, and 1% Triton X-100 at a pH of 8.0 and sonicated for three 1-min cycles at 4 °C. Inclusion bodies were pelleted at 20,000 × g for 30 min at 4 °C and washed twice with suspension solution without Triton X-100. Inclusion bodies were solubilized in a solution containing 50 mM Tris, 5 mM EDTA, and 8 M urea at a pH of 8.0 at room temperature overnight with agitation. After centrifugation, soluble protein was purified by chromatography through Ni-agarose. rOmp31 was adsorbed with Sepharose-polymyxin B to eliminate LPS contamination. Plasmid DNA for in vitro transfection or mouse immunization was extracted from a 16-h culture and purified using the Endo-Free Plasmid purification kit. Plasmid DNA was adjusted to a final concentration of 1 mg/ml in PBS and stored at −80 °C (Gupta et al., 2007) . Mice injected with pTargeTomp31 have good IgG2a and IgG1 titers to anti-omp31 antibodies. Antibodies against omp31 could already be detected 1 week after the first pTargeTomp31 DNA injection. Immunization with pTargeT did not induce any production of anti-omp31 antibodies. Humoral response measured 18 weeks postvaccination indicates that pTargeTomp31 DNA vaccine leads to the generation of long-lived IgG1 and IgG2a responses. pTargeTomp31 DNA vaccination resulted in specific T-cell proliferation in response to omp31 or to Brucella extracts. This specific induced proliferative response was also found at 8 weeks postvaccination, although to a lesser extent. In contrast, immunization with pTargeT appeared to have no effect on the level of T-cell proliferative response. The ConA mitogen was able to induce T-cell proliferation in all cases (Gupta et al., 2007). BALB/c Specific-pathogen-free 4-week-old BALB/c female mice were obtained. 25 mice were randomly divided in to five groups of 5 mice each and received intramuscular injections in the tibialis anterior muscles with 100 μg of pTargeTomp31 in 50 μl sterile saline by using a 1-ml insulin syringe with a 28-gauge needle. Similarly, another set of 6 weeks old 25 female mice (divided in five groups of 5 mice each) were sham immunized with saline only, the control group. From both the DNA immunized and sham immunized mice, one group of 5 mice each was exclusively used in order to obtain sera to test antibody and spleen cells for cytokine production in response to omp31 or B. melitensis extract. Three vaccinations at 3-week intervals were performed (Gupta et al., 2007). No significant difference in the number of the bacteria isolated from the spleens of the saline vaccinated and pTargeT-vaccinated animals was observed. The mice, which were immunized by pTargeTomp31, showed a significant level of protection at 28 days after challenge. Mice immunized with DNA vaccine made anti-Brucella serum antibody. This vaccine provided the moderate degree of protection to the mice (Gupta et al., 2007). Mice immunized three times with pTargeTomp31 or pTargeT were infected 9 weeks later the last DNA immunization with B. melietensis 16M. Seven, 14, 21 and 28 days after challenge, mice were killed and the number of bacteria in the spleens was quantitated (Gupta et al., 2007). B. melitensis bp26 and TF Nasal Vaccine VO_0000411 Recombinant vector vaccine Intranasal (i.n.) immunization with bp26 and Tf in conjunction with cholera toxin (CT) adjuvant (Yang et al., 2007). Recombinant bp26 and Tf were produced in Pichia pastoris and purified. Each vaccine consisted of either 50 or 100 micrograms per dose of either bp26, Tf, or both combined with the CT adjuvant (Yang et al., 2007). Purified Brucella bp26 and trigger factor (Tf) proteins, also called Tig (Yang et al., 2007). Recombinant protein preparation Recombinant bp26 and Tf were produced in Pichia pastoris and purified, and mixed with cholera toxin (CT) adjuvant(Yang et al., 2007). Mice immunized with Tf alone showed reduced serum and mucosal Ab titers. Between 3 and 5 weeks post-primary immunization, mice given bp26 + Tf showed between 14- and 32-fold greater serum IgG anti-Tf Ab titers when compared to mice given only Tf. Fecal IgA titers were augmented between 24- and 128-fold in mice immunized with bp26 + Tf when compared to mice immunized with Tf alone. These results clearly show that co-immunization with bp26 enhances anti-Tf immunity. To determine whether immune responses evoked by bp26 and Tf are biased towards Th1-type, Th2-type, or a mixture of both, serum samples from bp26 + Tf-immunized BALB/c mice from day 35 were evaluated for IgG subclass responses. IgG1 was the predominant IgG subclass Ab induced by the nasal immunization regimen. IgG2a and IgG2b anti-Tf and anti-bp26 Abs were also observed, but these were significantly less than the induced IgG1 Ab titers (Yang et al., 2007). BALB/c Specific pathogen-free female BALB/c mice were i.n. immunized with bp26 (50 μg/dose), Tf (100 μg/dose), or both + CT adjuvant on days 0, 7, and 14. Five μg of CT were given on day 0, and with subsequent boosts, 2 μg/dose were given. Mice were sampled for serum and fecal titers at 3–5 weeks post-immunization, before challenge (Yang et al., 2007). Mice immunized with bp26 and Tf exhibited significantly reduced B. melitensis colonization when compared to PBS-dosed control mice, the control. There was no significant difference between mice immunized with these proteins when compared to RB51-vaccinated mice. No significant differences in splenic weights were observed among any of the immunization groups. These findings show that nasal immunization with bp26 and Tf is able to reduce B. melitensis colonization, and the observed splenic inflammation may be contributed by CT immunization (Yang et al., 2007). Immunized mice were challenged i.p. with 5 × 10^4 CFU B. melitensis strain 16 M on day 28 post-primary immunization [13]. A positive vaccination control was given i.p. 1 × 10^8 CFU of B. abortus RB51 vaccine 8 weeks prior to challenge. Four weeks post-challenge, splenic CFU and splenic weights were determined (Yang et al., 2007). B. melitensis DNA vaccine encoding Omp31 boosted with Omp31 VO_0000436 DNA vaccine Research pCI-neo prime, recombinant protein boost [Ref489:Cassataro et al., 2005] Intramuscular injection (i.m.) A regimen of DNA prime and protein boost is followed, increasing the efficacy of the vaccine compared to DNA or protein alone (Cassataro et al., 2007). Incomplete Freund's Adjuvant For the propagation of plasmids, Escherichia coli strain JM109 was used. For the expression of the recombinant protein, strain BL21(DE3) was employed. Bacterial strains were routinely grown, and rOmp31 was obtained and treated as described by (Estein et al., 2003). Purity was determined by Coomassie blue staining as described by (Cassataro et al., 2005). The plasmid pCIOmp31 was obtained as described in (Cassataro et al., 2005). Intramuscular injection (i.m.) Omp31 protein DNA vaccine construction Immunoglobulin G1 (IgG1) and IgG2a titers were higher in sera from pCIOmp31/rOmp31-immunized mice than in sera from mice immunized with pCIOmp31 or rOmp31 alone. Splenocytes from pCIOmp31/rOmp31-immunized mice produced significantly higher levels of gamma interferon than did those from mice given rOmp31 alone. In contrast, interleukin 2 (IL-2) production levels were comparable between the two groups of immunized mice. Cells from all immunized mice produced undetectable levels of IL-4. Notably, rOmp31 stimulated IL-10 production in the pCIOmp31/rOmp31-immunized group but not in the pCIOmp31- or rOmp31-immunized group. Although the prime-boost regimen induced specific cytotoxic responses, these responses could not reach the levels achieved by the pCIOmp31 immunization (Cassataro et al., 2007). BALB/c mice was immunized with an Omp31 DNA vaccine (pCIOmp31) followed by boosting with recombinant Omp31 (rOmp31) in incomplete Freund's adjuvant (Cassataro et al., 2007). pCIOmp31 priming followed by rOmp31 boosting led to moderately improved protection against a challenge with B. ovis or B. melitensis (Cassataro et al., 2007). Immunized mice were challenged, by intravenous inoculation, with 1x10^5 B. melitensis H38S organisms or 1x10^5 B. ovis organisms. Mice were sacrificed 30 days after the bacterial challenge, and their spleens were removed, homogenized, plated, and incubated. The number of CFU per spleen or liver was counted, and the results were given as the mean log number of CFU (Cassataro et al., 2007). Cytokine secretion in culture supernatants of spleen cells from immunized mice was evaluated by ELISA 30 days after the last immunization. IFN-gamma levels were significantly higher in mice immunized with pCIOmp31 priming and rOmp31 boosting than mice immunized with PBS-, pCI-, or rOmp31 and boosted with rOmp31 (Cassataro et al., 2007). Cytokine secretion in culture supernatants of spleen cells from immunized mice was evaluated by ELISA 30 days after the last immunization. IL-2 levels were significantly higher in mice immunized with pCIOmp31 priming and rOmp31 boosting than mice immunized with PBS-, pCI-, or rOmp31 and boosted with rOmp31 (Cassataro et al., 2007). Cytokine secretion in culture supernatants of spleen cells from immunized mice was evaluated by ELISA 30 days after the last immunization. IL-10 levels were significantly higher in mice immunized with pCIOmp31 priming and rOmp31 boosting than mice immunized with PBS-, pCI-, or rOmp31 and boosted with rOmp31 (Cassataro et al., 2007). B. melitensis DNA vaccine p-ialB VO_0004397 DNA vaccine Research pcDNA3 [Ref863:Commander et al., 2007] Intramuscular injection (i.m.) Intramuscular injection (i.m.) DNA vaccine construction Vector pCR3.1 expressed IalB (Commander et al., 2007). Brucella specific IFNγ was still evident at 12 weeks post-vaccination, albeit at low concentration, indicating generation of a lasting Th1 immune response (Commander et al., 2007). VO_0000286 P-ialB vaccine was very promising in terms of control of Brucella challenge, as they were unable to recover viable Brucella from the spleens of 60% (three out of five) p-ialB treated mice of a study group following challenge with ∼1 × 104 CFU B. melitensis 16M per mouse. The remaining two mice showed significantly reduced Brucella loads compared to the unvaccinated (PBS and vector control) mice. If the data is considered in terms of the % of animals without detectable Brucella in their spleens (less than 10 CFU per spleen) at 15 days post-challenge, the effect of the p-ialB vaccination is judged to be equivalent to that of the Rev.1 live attenuated vaccine, with each vaccine able to ‘protect’ 60% of the test population. Moreover, a comparison of the total quantitative data available from each sample shows the Brucella load of the p-ialB and Rev.1 vaccinated animals to be significantly lower (p < 0.05, one-way ANOVA, Dunnett's post-test) than that observed for the unprotected controls (PBS or pcDNA3.1 vector control vaccinated mice). This vaccine was shown to have a protective index of greater than 2.5 (Commander et al., 2007). B. melitensis DNA vaccine p-Omp25 VO_0004396 DNA vaccine Research pcDNA3.1 [Ref863:Commander et al., 2007] Intramuscular injection (i.m.) Intramuscular injection (i.m.) DNA vaccine construction Vector pCR3.1 expressed Omp25 (25 kDa Omp) (Commander et al., 2007). Brucella specific IFNγ was still evident at 12 weeks post-vaccination, albeit at low concentration, indicating generation of a lasting Th1 immune response (Commander et al., 2007). VO_0000286 P-omp25 vaccine was very promising in terms of control of Brucella challenge, as researchers were unable to recover viable Brucella from the spleens of 60% (three out of five) p-omp25 treated mice of a study group following challenge with ∼1 × 104 CFU B. melitensis 16M per mouse. The remaining two mice showed significantly reduced Brucella loads compared to the unvaccinated (PBS and vector control) mice. If the data is considered in terms of the % of animals without detectable Brucella in their spleens (less than 10 CFU per spleen) at 15 days post-challenge, the effect of the p-omp25 vaccination is judged to be equivalent to that of the Rev.1 live attenuated vaccine, with each vaccine able to ‘protect’ 60% of the test population. Moreover, a comparison of the total quantitative data available from each sample shows the Brucella load of the p-omp25 and Rev.1 vaccinated animals to be significantly lower (p < 0.05, one-way ANOVA, Dunnett's post-test) than that observed for the unprotected controls (PBS or pcDNA3.1 vector control vaccinated mice). This vaccine was shown to have a protective index of greater than 2.5 (Commander et al., 2007). B. melitensis DNA vaccine pCI-Omp31 VO_0004394 DNA vaccine Research pCI [Ref489:Cassataro et al., 2005] Intramuscular injection (i.m.) Intramuscular injection (i.m.) DNA vaccine construction Vector pCI expressed Omp31 (hemin-binding protein) (Cassataro et al., 2005). The Omp31 DNA vaccine induces cytotoxic responses that have the potential to contribute to protection against Brucella infection. The protective response could be related to the induction of CD8(+) T cells that eliminate Brucella-infected cells via the perforin pathway (Cassataro et al., 2005). VO_0000286 Mice given pCIOmp31 exhibited a significant degree of protection against B. melitensis and B. ovis (P < 0.001) compared with controls receiving PBS. This was determined by comparing the levels of infection in the spleen (CFU) (Cassataro et al., 2005). B. melitensis DNA vaccine pCIBLSOmp31 encoding BLSOmp31 (fusion protein) VO_0004395 DNA vaccine Research pCI-neo [Ref222:Cassataro et al., 2007] Intramuscular injection (i.m.) Intramuscular injection (i.m.) DNA vaccine construction DNA vaccine construction The vaccine induced a strong humoral response against the inserted peptide. It also induced peptide- and BLS-specific cytotoxic T responses. The insertion of this peptide on BLS induced stronger T helper 1 responses specific for the carrier (BLS) (Cassataro et al., 2007). VO_0000286 BLSOmp31 (pCIBLSOmp31) provided the best protection level against Brucella ovis, which was significantly higher than the given by the co-delivery of both plasmids coding for the whole proteins (pcDNABLS+pCIOmp31) and even higher than the control vaccine Rev.1. Moreover, pCIBLSOmp31 induced higher protection against Brucella melitensis than pcDNABLS+pCIOmp31 (Cassataro et al., 2007). B. melitensis lipopolysaccharide (LPS) vaccine VO_0000311 subunit vaccine LPS, an outer membrane endotoxin common to gram negative bacteria, consists of three parts: Lipid A, a core oligosaccharide, and an O-antigen. The O-antigen is found in smooth strains only. None. The LPS was extracted from killed cells and purified by a modification of the method of Bundle et al (Bhattacharjee et al., 2006). B. melitensis strain 16M lipopolysaccharide (LPS) Groups of female BALB/c mice (Harlan Sprague-Dawley) were immunized by either i.n. or s.c. with purified B. melitensis LPS or LPS-GBOMP noncovalent complex vaccine. Control mice were immunized subcutaneously with sterile saline. Briefly, 25 µl of vaccine containing either 10 µg LPS or 10 µg LPS and 7.5 µg GBOMP contained in sterile saline was administered dropwise into the nostrils of anesthetized mice. Two doses of vaccine were given four weeks apart. For subcutaneous immunization, mice were given 10 µg vaccine in 200 µl sterile saline under the right hind thigh. A second dose of vaccine was given four weeks after the first dose. Blood was collected from five euthanized mice from each group four weeks after the first dose and four weeks after the second dose of vaccine. Sera were collected and stored at –20°C until analyzed for antibody. An enzyme-linked immunosorbent assay (ELISA) was used (Bhattacharjee et al., 2006). Mice immunized subcutaneously with LPS vaccine showed significant protection against infection of the spleen (P < 0.001), liver (P < 0.001), and lungs (P < 0.05). Groups of immunized mice (15 to 20 mice in each group) were challenged intranasally 4 weeks after the second dose of vaccine with 1x10^4 CFU of B. melitensis 16M suspended in 30 µl phosphate-buffered saline (PBS) (0.01 M sodium phosphate, 0.14 M sodium chloride; pH 7.5) as described previously (21). Blood, spleens, lungs, and livers were aseptically collected from anesthetized mice 8 weeks postchallenge. The numbers of Brucella CFU in organs were determined by dilution and culture on brucella agar as described previously (17). Serum was separated and stored at –20°C until it was used (Bhattacharjee et al., 2006). B. melitensis LPS-GBOMP VO_0000312 Subunit vaccine The outer membrane protein GBOMP (Bhattacharjee et al., 2002). The vaccines were stored at −20°C until use (Bhattacharjee et al., 2002). To create the vaccine, the LPS was extracted from killed B. melitensis cells and purified. N. meningitidis group B strain 8047 bacteria were grown in a synthetic medium, and outer membrane protein (GBOMP) was extracted. The B. melitensis LPS-GBOMP noncovalent complex vaccine was prepared with purified B. melitensis LPS dissolved in 42 ml of TEEN buffer. This mixture was added to the LPS solution and kept at 5°C for 30 min. The detergent was removed, and buffer was exchanged with sterile 0.9% NaCl solution. The final product was filtered and the LPS content was determined (Bhattacharjee et al., 2002). The antigens used in this vaccine were purified Brucella melitensis lipopolysaccharide (LPS) as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (GBOMP) (Bhattacharjee et al., 2002). Anti-B. melitensis LPS IgG titers were significantly increased two weeks after i.n. immunization with B. melitensis LPS-GBOMP vaccine. The pattern of IgG1 response closely resembled that of total IgG, but IgG2a and IgG2b responses were blunted compared to the IgG1 response,and the IgG3 response was also blunted compared to that of IgG1(Bhattacharjee et al., 2002). BALB/c Each mouse was given an intramuscular injection of 0.3 mg of xylazine hydrochloride and 1.0 mg of ketamine hydrochloride in 50 μl of sterile saline prior to immunization. The mice were then immunized by administration of 25 μl of vaccine containing 10 μg of LPS slowly into the nostrils with a micropipette, with a second dose of vaccine was given 4 weeks after the initial dose (Bhattacharjee et al., 2002). I.n. immunization with B. melitensis LPS-GBOMP subunit vaccine significantly protects mice against intranasal challenge with virulent B. melitensis. Vaccination reduces spleen and liver bacterial dissemination but has no effect on the course of lung infection (Bhattacharjee et al., 2002). Groups of immunized and control mice were challenged intranasally at various times with 10^4 CFU of B. melitensis 16 M suspended in 30 μl of phosphate-buffered saline. In one experiment, mice were challenged with 10^5 CFU of bacteria (Bhattacharjee et al., 2002). B. melitensis P39 deletion vaccine VO_0000633 Live, attenuated vaccine Research Intramuscular injection (i.m.) A B. melitensis vaccine that is recombinant B. melitensis Rev. 1 vaccine with the p39 gene deletion (Tibor et al., 1998). Intramuscular injection (i.m.) B. melitensis P39 protein vaccine VO_0000412 Subunit vaccine P39 has been found to be a Brucella protective antigen (Al-Mariri et al., 2001). A synthetic phosphorothioate oligodeoxynucleotide containing an unmethylated, consensus immunostimulatory CpG motif (5′-purine-purine-CpG-pyrimidine-pyrimidine-3′ oligodeoxynucleotide [CpG ODN]). Specifically, the immunostimulatory CpG 1826 (5′-TCCATGACGTTCCTGACGTT-3′) was used (Al-Mariri et al., 2001). Not reported. The bfr gene of Brucella melitensis 16M was subcloned and expressed in an expression vector. The protein was purified and mixed with the CpG ODN as the vaccine (Al-Mariri et al., 2001). Brucella melitensis 16M P39, a putative periplasmic binding protein (Al-Mariri et al., 2001). recombinant protein The bfr gene of Brucella melitensis 16M was subcloned into a pET-15b expression vector that contains a polyhistidine tag, and the resulting plasmid pET-15b-bfr was introduced in E. coli BL21(DE3). After 2 to 4 of induction with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside), bacterial cells from a 100-ml culture were washed once and then sonicated. The lysate was centrifuged for 10 min at 9,000 × g at 4°C. The pellet was kept frozen at −70°C. After it had thawed, the pellet was resuspended in a lysis buffer. The resulting lysate was centrifuged at 9,000 × g for 20 min at 4°C (Al-Mariri et al., 2001). P39 was then purified based on metal chelate affinity chromatography as described previously (Letesson et al., 1997). Balb/c mice Female BALB/c mice at 4 weeks of age were separated into nine groups of 12 mice. Groups 1, 2, and 3 received PBS, CpG ODN, and non-CpG ODN, respectively, and served as negative controls. Groups 4 and 5 were injected with the purified P39 and bacterioferrin (BFR) alone, respectively. Groups 6 and 7 were injected with the recombinant protein with CpG ODN adjuvant. Finally, groups 8 and 9 received the recombinant antigens with the non-CpG ODN. Vaccines were prepared in PBS and contained combinations of the following: 20 μg of recombinant protein and/or 20 μg of oligonucleotides when needed. Vaccines were given intramuscularly (i.m.) into the left tibial anterior muscles in a total volume of 50 μl three times at 3-week intervals (Al-Mariri et al., 2001). Not reported. None reported. Three weeks after the last injection, the remaining mice of each group were challenged by the intraperitoneal route (i.p.) with 5 × 10^4 CFU of B. abortus strain 544 in 100 μl of PBS. An additional group of eight mice vaccinated i.p. with B19 (1x10^5 CFU) was challenged 4 weeks later in the same way and served as a vaccinated control. Spleen colonization with the challenge strain was determined at 4 and 8 weeks postinfection (Al-Mariri et al., 2001). B. melitensis strain VTRM1 VO_0000300 attenuated live vaccine Vaccination with the rough strain VTRM1 induces protection against virulent strains of B. abortus (2308), B melitensis (16M), B. suis biovar 1 (750), B. suis biovar 4 (2579), and B. ovis (Winter et al., 1996). VTRM1, like RB51, is a stable rough form of B. melitensis that does not revert during passage in mice (Winter et al., 1996). VTRM1 was obtained from B. melitensis 16M by allelic exchange of the rfbU gene encoding mannosyltransferase with a Tn5-disrupted rfbU gene (Winter et al., 1996). VTRM1 Five groups of mice, (eight to nine weeks old), were vaccinated with the following live organism vaccines. These included: VTRM1, VTRS1, and Rev 1 (5 x 10^4 CFU); strain 19 (S19) (5 x 10^3 CFU); and RB51 (3 x 10^8 CFU). Each vaccine was delivered intravenously except RB51 which was administered intraperitoneally. All vaccines except RB51 were obtained directly from frozen stock cultures. Large doses of RB51 were required, therefore stock cultures were inoculated onto plates. After three days of culture and growth after 3 days was diluted to the appropriate concentration (Winter et al., 1996). Strains VTRM1 and VTRS1 were effective in inducing protection against virulent strains of heterologous, and homologous, Brucella spp. Vaccination with either strain conferred substantial protection against exposure to virulent laboratory strains of B melitensis, B abortus, and B suis of biovars 1 and 4. VTRM1 provided protection against B. ovis (Winter et al., 1996). Eight weeks after the initial vaccination, mice were injected IV with 5x10^4 CFU of Brucella. The time interval between IV injection and euthanasia was two weeks for all Brucella strains with the exception of B abortus 8-954 and 2-1230; for these strains, a one-week time interval was chosen. Mice were euthanized by CO2 asphyxiation. Individual spleens were homogenized, the homogenate diluted serially, and plated on Schaedler blood agar. B abortus, B melitensis, or B suis colonies were counted after three days of incubation; B. ovis colonies were counted after four to five days of incubation at 37 C under 10% CO2 (Winter et al., 1996). B. melitensis vaccine strain Rev. 1 VO_0000710 Attenuated live vaccine Licensed The live attenuated strain of B melitensis Rev . 1 is considered the best vaccine available for the prophylaxis of brucellosis in sheep and goats (Cloeckaert et al., 2002). The vaccine was kept on plates at 5 degrees celsius (ELBERG and FAUNCE, 1957). Whether Rev. 1 is virulent is unclear. A Rev. 1-like strain was isolated from the membranes of an aborted fetus at an intensively managed farm that had complied with the vaccination regimen, i.e. ewelambs were vaccinated between two and six months. When the whole flock (410 ewes) was serologically tested, 34 animals showed a positive complement fixation test. The ewes were slaughtered and attempts to culture additional isolates from milk, major lymph nodes, and mammary glands were unsuccessful. A few months later , the flock owner contracted brucellosis and Brucella was cultured from his blood (Banai, 2002). Rev . 1 vaccine, obtained in the 1950s by a two-step selection involving firstly, a streptomycin resistance and/or dependence, and secondly, a reversion of dependence but maintaining streptomycin resistance (ELBERG and FAUNCE, 1957). Acquired chromosomal streptomycin resistance is frequently due to mutations in the gene encoding for ribosomal protein S12, rpsL (Cloeckaert et al., 2002). Rev.1 is a live attenuated Brucella melitensis strain derived from a virulent B. melitensis isolate. Recombinant protein preparation The gene rpsL of B. melitensis reference strain 16 M encodes for ribosomal protein S12. This gene was mutated in the vaccine strain Rev. 1 during its natural selection process. Nucleotide sequencing has revealed that a mutation in the rpsL gene of vaccine strain Rev . 1 compared to that of reference strain 16M leading to an amino acid Pro- to -Leu change at codon position 91 ( Pro91Leu ) (Cloeckaert et al., 2002). Several groups of 10 (or eight mice in the assay after storage or passage) were vaccinated subcutaneously with Rev. 1 strains. Control unvaccinated mice received BSS alone (Bosseray, 1991). Vaccinated and control mice were challenged intraperitoneally 30 or 45 days after vaccination with The virulent B. abortus 544 CO2-dependent reference challenge strain (Bosseray, 1991). Seven groups, 22 sheep/group were either vaccinated subcutaneously (SC) or conjunctivally (CJ) (right eye) at the age of four months with the following actual doses, Rev.1, 1.1 × 10^9 CFU (SC) and 1.33 × 10^9 CFU (CJ); CGV26, 1.25 × 10^9 CFU (SC) and 1.22 × 10^9 cfu (CJ); CGV2631, 1.11 × 10^9 cfu (SC) and 1.30 × 10^9 cfu (CJ). The vaccine doses (<1 mL) were utilized where administered SC and 30 uL where administered CJ (Jacques et al., 2007). Regardless of the route of administration of Rev.1 vaccine (SC or CJ), the results obtained agreed with those of previous studies [35], Rev.1 effectively protected ewes challenged at middle of pregnancy with 5 × 10^7 CFU of strain H38. In the control group (unvaccinated ewes), 100% aborted (Jacques et al., 2007). One week before challenge, of 154 ewes, 99 of whiche were pregnant assesd by progesterone levels were were kept in a high security pen. Each ewe was challenged CJ (left eye) with 5.1 × 10^7 CFU of B. melitensis strain H38 after 76 days of pregnancy. Each animal was submitted to periodic immunological, clinical, and bacteriological tests and sacrificed 4–6 weeks after delivery. To follow cellular response, six ewes from each group were selected for further investigation (Jacques et al., 2007). The mutant strain stimulated an antibody-producing mechanism within the first ten days of vaccination. The antibody titers were highest at about 20 days postinfection (ELBERG and FAUNCE, 1957). Angora Four to five-year-old goats were used. Each goat received 1.5 x 10^9 cells of the bacteria subcutaneously in the left prescapular region. At 2,4,5,7,10,11, and 14 weeks the goats were sacrificed and tested for the presence of brucellae (ELBERG and FAUNCE, 1957). At the third week organisms from the spleen, bone marrow, left and right prescapular regions, left and right supramammary nodes, right precrural and the mediastinal nodes were detected. By the fourteenth week, the infection in the single goat sacrificed was isolated to the left prescapular node. It was assumed that at this point the animals were free from infection (ELBERG and FAUNCE, 1957). The vaccine was proven to be an effective immunizing agent for the host species tested (ELBERG and FAUNCE, 1957). The challenge consisted of 33 ID doses of B. melitensis. It was administered 90 days after vaccination (ELBERG and FAUNCE, 1957). B. melitensis with deleted bp26 (CGV26) VO_0000338 Live, attenuated vaccine The virulence of the CGV26 mutant was similar to that of the parental Rev.1 strain (Guilloteau et al., 2006). Mutants were kept freeze-dried. Bacteria were rehydrated in sterile buffered saline solution, seeded on Trypticase Soy Agar, supplemented with yeast extract and incubated at 37 °C. Fresh bacterial suspensions were harvested in BSS from 24 h cultures and spectrophotometrically adjusted. Each suspension used was always checked for purity and dissociation and the exact doses were assembled (Guilloteau et al., 2006). The antigen for this vaccine is Brucella melitensis containing a deletion of the bp26 gene, a strain called CGV26 (Guilloteau et al., 2006). Recombinant protein preparation The CGV26 and CGV2631 strains were engineered with Brucella melitensis Rev.1 mutant strains containing deletions in the bp26 gene or both bp26 and omp31 genes (Guilloteau et al., 2006). The results together showed that the CGV26 mutant was able to induce both specific antibody response and systemic lymphoproliferation in sheep. Specific systemic antibody response to S-LPS of Brucella was induced after inoculation of the mutant and the Rev.1 strain. The intensity of this response was much higher and earlier after SC vaccination (1 week) than that after CJ vaccination (second week) (Guilloteau et al., 2006). Préalpes 42 Brucella-free Préalpes females of 3–4 months of age were randomly allotted into seven groups of six animals. Six groups were inoculated either by CJ or SC route with 1 × 10^9 cfu of CGV26 mutant or the Rev.1 vaccine as a vaccinated control group. The vaccine suspension was administered conjunctivally with a calibrated dropper by depositing 33 μl on the surface of the right eye or subcutaneously with a needle by injecting 1 ml just behind the left elbow joint. One group remained as a separate uninfected control group. Swabs were taken from the conjunctiva and nostrils in groups inoculated by CJ route for bacteriological analyses. At regular intervals after inoculation, blood samples were collected for analysis of the immune response induced (Guilloteau et al., 2006). The local persistence of the CGV26 mutant was not statistically different from the Rev.1 strain (Guilloteau et al., 2006). This mutant induced significant specific antibody and cell-mediated immunity in sheep (Guilloteau et al., 2006). B. melitensis WR201 (16MΔpurEK) VO_0000345 Live, attenuated vaccine The antigens for this vaccine were severely attenuated. The strain WR201 fails to replicate in cultured human monocyte-derived macrophages (Hoover et al., 1999). The WR201 strain was procured and the bacterial cells were killed by treatment for 16 h with 0.5% phenol. These cells were then washed and pelleted several times, eventually leading to a product containing approximately 3.0 mg of protein per ml (Hoover et al., 1999). This vaccine is an live, attenuated purine-auxotrophic mutant strain of Brucella meletensis, WR201 (Hoover et al., 1999). Recombinant protein preparation The B. melitensis strain WR201 (or called delta purE201) was created through the deletion of the gene purE and the deletion of the first seven bases of purK (Drazek et al., 1995)(Hoover et al., 1999). Specifically, B. melitensis 16M was electroporated with suicide plasmids containing a kanamycin resistance cassette that replaced 226 bp at the carboxyl end of purE, the intergenic region, and 18 bases of the purK open reading frame. Recombinant B. melitensis delta purE201 required exogenous purines for growth on minimal media. This mutant failed to grow in human monocyte-derived macrophages, while the growth of wild-type 16M and the complemented strain, delta purE201 (pSD5), increased by nearly two logs (Drazek et al., 1995). Gene mutation This purE mutant is from Brucella melitensis (Crawford et al., 1996). After mice were innoculated, they were found to have made serum antibody to non-O-polysaccharide and lipopolysaccharideantigens. The splenocytes from the immunized animals were found to have released interleukin-2, gamma interferon, and IL-10 when cultured with Brucella antigens (Hoover et al., 1999). BALB/c The mice were immunized by intraperitoneal administration of vaccine. Each vaccine contained approximately 10^5 WR201 cells Nonimmunized, control mice received 0.9% NaCl intraperitoneally (Hoover et al., 1999). Immunization of the mice led to protection from disseminated infection. Immunization had only a slight effect on the clearance of the challenge inoculum from the lungs (Hoover et al., 1999). Nine weeks post immunization, animals were innoculated intranasally with 10^4 CFU of B. melitensis 16M in 30 μl of 0.9% NaCl. The vaccine was administered dropwise into the external nares with a micropipette (Hoover et al., 1999). This study suggests that WR201 may be a good vaccine candidate for the prevention of human brucellosis (Hoover et al., 1999). Spleen cells obtained 9 weeks after inoculation of mice with WR201 produced more IL-2 than non-immunized, non-infected control mice. These results were significant (Hoover et al., 1999). Spleen cells obtained 9 weeks after inoculation of mice with WR201 produced more IL-10 than non-immunized, non-infected control mice. These results were significant (Hoover et al., 1999). Spleen cells obtained 9 weeks after inoculation of mice with WR201 produced more IFN-gamma than non-immunized, non-infected control mice. These results were significant (Hoover et al., 1999). Sera obtained from immunized animals from 1 to 8 weeks after intraperitoneal administration of WR201 showed a rise in anti-protein IgG by week 4 (Hoover et al., 1999). When grown in cultures with Brucella antigens, splenocytes from immunized animals released interleukin-2 and gamma interferon. Immunized mice made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens (Izadjoo et al., 2004). BALB/c Mice were first orally administered 0.2 ml of sterile 2.5% sodium bicarbonate, then they were administered 0.2 ml of the bacterial suspension which had been standardized to 5 × 10^11 cells/ml. In designated experiments, bacteria were further diluted to provide an inoculum of 1010 or 109 CFU, then administered. In another experiment, strain WR201 was killed by treatment overnight at room temperature with 0.8% (vol/vol) formaldehyde prior to administration to mice (Izadjoo et al., 2004). No bacteria were recovered from the spleens of 54 mice eight weeks post-vaccination, while no bacteria were recovered from the iguinal lymph nodes of 15 mice at this time either. Strain WR201 was progressively lost from the feces following oral administration (Izadjoo et al., 2004). The mice that were immunized had protection from infection and better clearance of the challenge inoculum from the lungs. The best protection was found with the administration of live bacteria with the inclusion of a booster dose. The results suggest that strain WR201 may be a candidate for a vaccine to prevent human brucellosis (Izadjoo et al., 2004). For virulent strain 16M i.n. challenge, 30 μl of bacterial suspension adjusted to contain 104 CFU of bacteria. This was administered with a micropipette into the external nares of mice (Izadjoo et al., 2004). B. suis strain 2 VO_0000722 Live, attenuated vaccine Licensed Intramuscular injection (i.m.) China An orally administrable brucellosis vaccine that was was developed in China. It is effective for oral vaccination of sheep, goats, cattle and pigs (Xin, 1986). Intramuscular injection (i.m.) B. suis strain VTRS1 VO_0000303 Attenuated live vaccine Vaccination with the rough strain VTRS1 induces protection against virulent strains of B. abortus (2308), B melitensis (16M), B. suis biovar 1 (750), B. suis biovar 4 (2579) (Winter et al., 1996). VTRS1, like RB51, is a stable rough form of the bacterium. It does not manifest evidence of reversion during passage in mice (Winter et al., 1996). The rough mutant VTRS1 was obtained from B. suis 2579 by allelic exchange of the rfbU gene encoding mannosyltransferase with a Tn5-disrupted rfbU gene (Winter et al., 1996). Mice, (eight to nine weeks old), in experimental groups of five, were vaccinated with live vaccines: VTRM1, VTRS1, and Rev 1 with 5 x 10^4 CFU (IV); strain 19 (S19) 5 x 10^3 CFU given IV; and RB51, 3 x 10^8 CFU given i.p. All vaccines except RB51 were obtained from frozen stock cultures. The large dose of RB51 administered required that stock cultures be cultured on plates, and after three days diluted to the appropriate concentration (Winter et al., 1996). VTRM1 and VTRS1 were effective in inducing protection against virulent strains of heterologous, as well as the homologous, Brucella spp. Vaccination with either strain conferred protection against challenge exposure with virulent laboratory strains of B. abortus, B. melitensis, and B. suis of biovars 1 and 4. VTRM1 also provided protection against B. ovis (Winter et al., 1996). Eight weeks after the initial vaccination, mice were challenged IV with 5x10^4 CFU of Brucella. From challenge exposure to euthanasia was 2 weeks for all Brucella strains except B abortus 8-954 and 2-1230. For these strains a 1-week interval was used. Mice were euthanized with CO2. Individual spleens were homogenized, diluted serially, and plated on Schaedler blood agar. Colonies were counted after incubation for 3 days (B. abortus, B. melitensis, and B. suis) or 4 to 5 days (B. ovis) at 37 C under 10% CO2 (Winter et al., 1996). Brucella abortus bacA mutant VO_0000347 Live, attenuated vaccine The bacA mutant strain designated as bacAmut-KL7 was produced by deleting a portion of the bacA gene from the parent strain, B. abortus 2308. The vaccines were produced, each dose containing 5 × 10^4 bacterial colony forming units per dose (Parent et al., 2007). The antigen for this vaccine is a bacA deletion mutant of Brucella abortus, known as KL7 (bacAmut-KL7) (Parent et al., 2007). The C57BL/6 mice mounted a strong TH1 immune response to infection with B. abortus which was characterized by continuous IFNγ production. At 4 weeks post-infection, a time corresponding to the plateau phase of infection, there was no significant difference in the number of 2308 and bacAmut-KL7 CFU recovered from C57BL/6 mice. BacAmut-KL7 was significantly attenuated in BALB/c mice during this time. During the clearance phase of the infection bacAmut-KL7 did show significant attenuation in C57BL/6 mice relative to 2308 although it was much less than that observed in BALB/c mice (Parent et al., 2007). BALB/c, C57BL/6 and C57BL/6 IFNγ−/− mice Eight to 12-week-old mice were infected intra-peritoneally with 5 × 10^4 bacterial colony forming units of B. abortus 2308, bacAmut-KL7 or hfq3 (Parent et al., 2007). IFNγ-activated macrophages equivalently controlled strains 2308 and bacAmut-KL7. The bacAmut-KL7 organism and its LPS induced greater amounts of pro-inflammatory cytokines than 2308 (Parent et al., 2007). The bactAmut-KL7 mutant was more pathogenic in C57BL/6 interferon-γ-deficient mice than 2308 causing abscesses and wasting even though the splenic loads of bacAmut-KL7 were significantly lower (Parent et al., 2007). No challenge was conducted, but it is known that the mutant created, bacAmut-KL7, has protected against challenge in previous experiments (Parent et al., 2007). Brucella abortus exsA mutant vaccine VO_0002812 Live, attenuated vaccine Research Intraperitoneal injection (i.p.) Intraperitoneal injection (i.p.) Gene mutation This exsA mutant is from Brucella abortus (Rosinha et al., 2002a). Brucella DeltaexsA mutant showed decreased survival in mice compared to the survival of parental strain S2308 (Rosinha et al., 2002a). Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51 (Rosinha et al., 2002a). Brucella abortus pgk mutant vaccine VO_0002818 Live, attenuated vaccine Research Intraperitoneal injection (i.p.) Intraperitoneal injection (i.p.) Gene mutation This pgk mutant is from Brucella abortus strain 2308 (Trant et al., 2010). A pgk mutant is attenuated in mice (Trant et al., 2010). A pgk mutant induces significant protection from challenge with wild type B. abortus (Trant et al., 2010). In IRF-1 KO mice, significantly increased levels of IFN-gamma were produced after vaccination with a pgk mutant as compared to medium. Animals immunized with strain RB51 showed reduced production of IFN-γ compared to mice vaccinated with the Δpgk mutant and S19, but it was not a statistically significant difference (Trant et al., 2010). Brucella abortus pgm mutant vaccine VO_0002819 Live, attenuated vaccine Research Intraperitoneal injection (i.p.) Intraperitoneal injection (i.p.) Gene mutation This pgm mutant is from Brucella abortus (Ugalde et al., 2003). A pgm mutant is attenuated in mice (Ugalde et al., 2003). A pgm mutant induces significant protection in mice from challenge with wild type B. abortus (Ugalde et al., 2003). Spleen cells from Δpgm-vaccinated mice were induced to secrete high levels of IFN-γ 8 weeks after immunization after stimulation with heat-inactivated B. abortus S2308 whole cells. The levels were compared to a positive control, the nonspecific mitogen ConA, and were significant (Ugalde et al., 2003). Brucella abortus S19 vjbR mutant vaccine VO_0004283 Live, attenuated vaccine Research Intraperitoneal injection (i.p.) Intraperitoneal injection (i.p.) Gene mutation The vjbR was mutated in Brucella abortus strain 19 (Arenas-Gamboa et al., 2009). An encapsulated vjbR mutant is attenuated in mice (Arenas-Gamboa et al., 2009). A single dose of an encapsulated vjbR mutant induced protection in mice from challenge with wild type Brucella (Arenas-Gamboa et al., 2009). Mouse immunization with encapsulated S19 ΔvjbR::Kan induced higher IgG1 and IgG2a levels compared to the nonencapsulated S19 ΔvjbR::Kan mutant 3 weeks post vaccination and significantly higher levels than naive mice (Arenas-Gamboa et al., 2009). Mouse immunization with encapsulated S19 ΔvjbR::Kan induced higher IgG1 and IgG2a levels compared to the nonencapsulated S19 ΔvjbR::Kan mutant 3 weeks post vaccination and significantly higher levels than naive mice (Arenas-Gamboa et al., 2009). Brucella Abortus Strain 19, Live Culture Vaccine (USDA: 1251.01) Colorado Serum Company VO_0001575 Live vaccine Licensed USA Brucella Abortus Strain 19, Live Culture, Reduced Dose Vaccine (USDA: 1251.02) Colorado Serum Company VO_0001576 Live vaccine Licensed USA Brucella Abortus Strain RB-51, Live Culture Vaccine (USDA: 1261.00) Colorado Serum Company VO_0001577 Live vaccine Licensed USA Brucella DNA vaccine encoding chimera BLSOmp31 VO_0001144 DNA vaccine pCI-neo [Ref222:Cassataro et al., 2007] Brucella outer membrane protein and lumazine synthase from Brucella spp. (BLS) induce protection against Brucella spp. A novel mode of delivering these antigens was devised as a DNA vaccine (Cassataro et al., 2007). None. To develop the chimerical plasmid, the BLSOmp31 DNA sequence was amplified by PCR using pETBLSOmp31 as template and sub-cloned in the pCI-neo vector (Promega, Madison, WI). The following oligonucleotides were constructed including restriction sites at the 5 Brucella outer membrane protein and lumazine synthase from Brucella spp. (BLS) (Cassataro et al., 2007). Mice were anaesthetized and immunized by the intramuscular route with 100 ug of pcDNABLS, pCIOmp31, pCIBLSOmp31, pcDNABLS + pCIOmp31 (100 Vaccinationof BALB/c mice with pCIBLSOmp31 provided optimal protection level against B. ovis (3.14 log), a value significantly higher than the protection elicited by the co-delivery of two plasmids (pCIOmp31+pcDNABLS) (2.20log) or by the Rev.1 vaccine (2.42 log). Mice vaccinated with pCIOmp31 exhibited a significant protection (2.24 log) against B. ovis; pcDNABLS induced 1.94log units of protection. pCIBLSOmp31 induced significantly higher protection (1.77log) against B. melitensis than pCIOmp31 plus pcDNABLS (1.09log) and similar protection than Rev.1 (2.30 log). Mice given pCIOmp31 exhibited a significant degree of protection(1.44 log) against B. melitensis as was against B. melitensis (1.44 log), while pcDNABLS induced 1.22 log protection. Together these results show that the chimera significantly increases protection elicited against B. ovis with respect to either pcDNABLS, pCIOmp31 or Rev.1 and induces a similar degree of protection against B. melitensis infection than Rev.1 vaccination. Thirty days after the last DNA injection, mice were challenged with 1 ×10^4 CFU of B. melitensis H38S or B. ovis (i.v.). Thirty days post challenge the animals were sacrificed and their spleens aseptically removed. Brucella melitensis bp26 mutant vaccine VO_0004000 Live, attenuated vaccine Research subcutaneous injection subcutaneous injection Gene mutation This bp26 mutant is from Brucella melitensis (Cloeckaert et al., 2004). A bp26 mutant is attenuated in mice (Cloeckaert et al., 2004). A bp26 mutant induces significant protection in mice from challenge with wild type B. melitensis (Cloeckaert et al., 2004). Brucella melitensis mucR mutant vaccine VO_0004277 Live, attenuated vaccine Research Intramuscular injection (i.m.) Intramuscular injection (i.m.) Gene mutation This gene is mutated in the B. melitensis 16M mucR mutant (Arenas-Gamboa et al., 2011). A mucR mutant has a significantly reduced degree of colonization in mice (Arenas-Gamboa et al., 2011). A mucR mutant of 16M protected against challenge with wild type B. melitensis 16M (Arenas-Gamboa et al., 2011). Brucella melitensis omp25 mutant vaccine VO_0004001 Live, attenuated vaccine Research Intravenous injection (i.v.) Intravenous injection (i.v.) Gene mutation This omp25 mutant is from Brucella melitensis (Edmonds et al., 2002). An omp25 mutant is attenuated in mice (Edmonds et al., 2002). An omp25 mutant induces protection from challenge with wild type Brucella melitensis (Edmonds et al., 2002). Brucella melitensis omp31 mutant vaccine VO_0004002 Live, attenuated vaccine Research subcutaneous injection subcutaneous injection Gene mutation This omp31 mutant is from Brucella melitensis (Cloeckaert et al., 2004). An omp31 mutant is attenuated in mice (Cloeckaert et al., 2004). An omp31 mutant induces significant protection in mice from challenge with wild type B. melitensis (Cloeckaert et al., 2004). Brucella ovis Microparticle Subunit Vaccine VO_0004146 Subunit vaccine The antigenic extract (HS) was obtained from B. ovis REO 198 cells. The bacterial cells were cultured, suspended in distilled water, and heat-killed in flowing steam. Following centrifugation, the supernatant was dialyzed against deionized water. The dialyzed material was ultracentrifuged, and the pellet (HS) was washed in dH2O and freeze-dried. The batch of antigen used to prepare the vaccine formulation contained 48.7 ± 5.0% protein and 41.7 ± 4.7% rough lipopolysaccharide (R-LPS). The vaccine consisted of F68–CD–MP microparticles suspended in saline. F68,CD, and MP are different excipients that were used in order to facilitate the encapsulation and conserve the bioactivity of the encapsulated antigenic complex (HS) (Estevan et al., 2006). A high hydrophobic antigenic complex taken from Brucella ovis (HS) (Estevan et al., 2006). BALB/c Five groups of five female BALB/c mice, 9 weeks old, were immunized subcutaneously with one of the following treatments: (i) F68–CD–MP microparticles containing 20 μg of HS per mouse in 0.1 mL saline; (ii) free HS (20 μg/mouse); (iii) empty TROMS microparticles; (iv) 5 × 10^4 CFU/mouse of the living attenuated B. melitensis Rev1 reference vaccine strain; or (v) sterile buffered saline solution, the unvaccinated control group (Estevan et al., 2006). A single dose administered s.c. 8 weeks before infection produced significant protection with respect to unvaccinated control mice. Moreover, this protection was similar to that conferred by B. melitensis Rev1 reference vaccine. In contrast, no significant protection was obtained when non-encapsulated HS was given to mice (Estevan et al., 2006). Eight weeks after vaccination, the mice were challenged by the intraperitoneal route with 1 × 10^5 CFU of B. melitensis H38 virulent strain. Two weeks after the challenge, the animals were euthanatised by cervical dislocation. The spleens of the animals were aseptically removed, individually homogenized in BSS, properly diluted, and plated in Blood Agar Base medium for viable counts (Estevan et al., 2006). Brucella recombinant SurA protein vaccine VO_0000358 Subunit vaccine Research Nasal spray, Intraperitoneal injection (i.p.) The PCR primers for cloning SurA gene from B. abortus were as follows: sense 5′AGAAAGCATATGTTTGCAAGACCTCTT3′ (NdeI) and antisense 5′TCTTCGGGATCCTCAACGATTGACGATGGT3′ (BamHI). B. abortus genomic DNA was used as template for PCR with Pfu DNA polymerase (Stratagene). The surA gene was cloned to plasmid Pet17b and then transformed to E. coli JM19 and then BL21(DE3) competent cells. Recombinant proteins were adsorbed with Sepharose-polymyxin B (Sigma, St. Louis, MO) to eliminate lipopolysaccharide contamination. The antigen and PBS were administered mixed with Complete Freund's Adjuvant (CFA) (Sigma) on day 0 and with Incomplete Freund's Adjuvant (IFA) on day 15 (Delpino et al., 2007). Nasal spray, Intraperitoneal injection (i.p.) The antigen for this vaccine is SurA protein from B. abortus strain 2308, B. abortus strain S19, and Brucella melitensis strain H38 (Delpino et al., 2007). Recombinant protein preparation Mice given rSurA with adjuvant exhibited a significant degree of protection against B. abortus infection (Delpino et al., 2007b). Immunization with rSurA elicited a vigorous IgG response that was detectable after the first immunization, increased further after the second Ag injection. PBS-immunized animals challenged with B. abortus 2308 developed antibodies against rSurA at 1 month after infection. Anti-rSurA IgG2a titers were higher than IgG1 titers during the whole immunization. rSurA stimulated significant production of IFN-γ, IL-2, IL-4 and IL-5 in spleen cells from rSurA-immunized animals but not from the PBS control group. All animals immunized with rDnaK alone elicited a humoral immune response that was detectable 15 days after the first immunization and increased further after the second injection to reach an IgG mean titer of 217,000 at day 30 post-vaccination. Immunization with rDnaK plus adjuvant induced similar anti-rDnaK IgG titers than immunization with rDnaK alone. None of the animals inoculated with PBS showed specific anti-rDnaK Abs at the time of challenge but notably, 30 days after infection all of them produced anti-rDnaK. Stimulation with rDnaK induced a significant production of IFN-γ and IL-2 in spleen cells from all mice immunized with rDnaK plus adjuvant Cells from rDnaK alone- or PBS-immunized mice were unable to stimulate the secretion of IFN-γ, IL-2, IL-4, IL-5 or IL-10 in response to rDnaK (Delpino et al., 2007). Balb/c Female BALB/c mice of 6 to 8-weeks old were used for the testing of these vaccines. Mice were anaesthetized with methoxyfuorane and immunized by the intraperitoneal route with (i) 30 μg of rSurA, (ii) 30 μg of rDnaK, (iii) rSurA + rDnaK (30 μg + 30 μg) or iv) PBS, which was the negative control. Antigens and PBS were administered mixed with Complete Freund's Adjuvant (CFA) on day 0 and with Incomplete Freund's Adjuvant (IFA) on day 15. A fifth group of mice was immunized i.p with 30 μg rDnaK in PBS without no adjuvant on days 0 and 15. As reference vaccinated controls other groups were immunized once (i) by the subcutaneous route at day 0 with 8 × 10^8 formalin-killed B. melitensis H38S in IFA or (ii) i.p with 1 × 10^4 live B. abortus S19. Two separate assays of immunization were performed. The first experiment included groups immunized with rDnaK plus adjuvant, rDnaK without adjuvant, rSurA, and the negative (PBS) and reference (H38) control groups. The second experiment included groups immunized with rDnaK, rSurA, and rDnaK + rSurA, all with adjuvant, and the negative (PBS) and reference (B. abortus S19) control groups. Sera for antibody detection were obtained by retro-orbital bleeding under anaesthesia at 15, 30, 45 and 75 days after the first immunization (Delpino et al., 2007). Mice given rSurA or rDnaK plus adjuvant exhibited a significant degree of protection against B. abortus when compared with controls receiving PBS. Formalin-killed B. melitensis H38S, the control vaccine, induced 2.19 units of protection against B. abortus. Immunization with rDnaK alone induced a low but still significant level of protection. In a second experiment, both evaluated vaccines (rDnaK or rSurA plus adjuvant) induced significant protection against B. abortus infection. There was no additive protection by the simultaneous immunization with both rDnaK and rSurA. All evaluated vaccines induced less protection than H38 or B. abortus strain 19 control vaccines. Altogether these results indicate that rSurA or rDnaK in adjuvant induce partial protection against B. abortus infection (Delpino et al., 2007). Immunized mice were challenged by i.p. injection with 1 × 10^4 B. abortus 2308 (Delpino et al., 2007). Spleen cells from mice were used to determine cytokine levels 1 month after last immunization. Mice immunized with rSurA and stimulated with SurA had significantly higher levels of IFN-gamma than did mice immunized with PBS and stimulated with SurA (Delpino et al., 2007b). Spleen cells from mice were used to determine cytokine levels 1 month after last immunization. Mice immunized with rSurA and stimulated with SurA had significantly higher levels of IL-2 than did mice immunized with PBS and stimulated with SurA (Delpino et al., 2007b). Spleen cells from mice were used to determine cytokine levels 1 month after last immunization. Mice immunized with rSurA and stimulated with SurA had significantly higher levels of IL-4 than did mice immunized with PBS and stimulated with SurA (Delpino et al., 2007b). Spleen cells from mice were used to determine cytokine levels 1 month after last immunization. Mice immunized with rSurA and stimulated with SurA had significantly higher levels of IL-5 than did mice immunized with PBS and stimulated with SurA (Delpino et al., 2007b). CGV2631 B. melitensis Rev. 1 with bp26 and omp31 deletions VO_0001171 Live, attenuated vaccine Research Intramuscular injection (i.m.) Intramuscular injection (i.m.) B. melitensis Rev. 1 with bp26 and omp31 deletions (Jacques et al., 2007). Sheep were conjunctivally or subcutaneously vaccinated at 4 months old (Jacques et al., 2007). eletion of bp26 and omp31 genes did not significantly affect the well recognised capacity of Rev.1 to protect sheep against B. melitensis challenge. However, the protection conferred by the CGV2631 mutant was significantly lower than that conferred by the CGV26 mutant or the Rev.1 strain (Jacques et al., 2007). Sheep were challenged with B. melitensis H38 at the middle of the first pregnancy following vaccination (Jacques et al., 2007). Divalent DNA B. abortus Vaccine pcDNA3.1-L7/L12-Omp16 VO_0000374 DNA vaccine pcDNA3.1(+) [Ref697:Luo et al., 2006b] Full-length open reading frames of the L7/L12 gene and Omp16 gene were amplified with PCR from the genome of B. abortus strain RB51. The gene amplified with L7/L12 primers and/or the gene amplified with Omp16 primers were inserted into pcDNA3.1(+) vector (Invitrogen) (Luo et al., 2006b). Brucella abortus L7/L12 protein and Omp16 protein were the antigens used in this vaccine. The vaccine was designated as pcDNA3.1-L7/L12-Omp16 (Luo et al., 2006b). divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. DNA vaccine construction DNA vaccine construction The results showed that the total IgG titer of the hyperimmune sera from mice immunized with pcDNA3.1-L7/L12-Omp16, pcDNA3.1-L7/L12, or pcDNA3.1-Omp16 reached 1:1,000, 1:800, or 1:600, respectively. Immunization with rL7/L12 and live RB51 strain elicited much higher humoral immune responses in mice, with IgG titers reaching 1:25,600 and 1:50,000, respectively. The analysis of IgG subtypes showed a significant increase in IgG1 and IgG2a from the DNA vaccine group, fusion protein vaccine group, and live RB51 group compared with the pcDNA3.1 vector control (Luo et al., 2006b). BALB/c Female BALB/c mice were distributed into two groups: each mouse in one group was inoculated intramuscularly with 100 μg of pcDNA3.1-L7/L12-Omp16, pcDNA3.1-L7/L12, or pcDNA3.1-Omp16 in 100 μl of PBS. A control group was also used in this group, and these mice were infected with PBS or the pcDNA3.1 expression vector alone. Each mouse in the other group was injected with 10 μg of rL7/L12-Omp16 in 100 μl PBS according to the same schedule. Every mouse in this group was injected on weeks 0, 2, and 4. The mice used as positive controls were inoculated intraperitoneally on day 0 with 2× 10^8 CFU of B. abortus strain RB51 in 0.2 ml of PBS (Luo et al., 2006b). Immunization with any type of DNA vaccine resulted in a significantly higher degree of protection than the controls that received only PBS. Within the three recombinant DNA vaccines, the divalent DNA vaccine provided a higher protection level than either of the univalent DNA vaccines. Immunization with the recombinant fusion protein rL7/L12 also created significant protection. This protective effect was significantly lower than that created by theimmunization with the DNA vaccines (Luo et al., 2006b). Five mice from each group were challenged intraperitoneally 14 days after initial immunization. These mice were given a higher dose of strain 544, 5 × 10^5 CFU (Luo et al., 2006b). E. coli Escheriosome-mediated Cytosolic Delivery of recombinant Brucella rL7/L12 Protein VO_0000423 Subunit vaccine Complete Freund’s Adjuvant (Mallick et al., 2007). The rL7/L12 proteins were purified and introduced into Escherischia coli lipid liposome, allowing for escheriosome-mediated cytosolic delivery of the antigen, recombinant rL7/L12 protein. Combinations containing rL7/L12 protein and different adjuvants were formed for vaccination (Mallick et al., 2007). The vaccine antigen is Brucella L7/L12 protein, which was delivered by an E. coli lipid liposome (escheriosome)-mediated cytosolic delivery system (Mallick et al., 2007). Analysis of the mouse sera revealed non-significant antibody titre up to day 14 post-immunization in various groups of immunized mice. Significantly higher titre of IgG was detected on day 21 post-immunization in sera of animals which were primed with E-Lip-L7 form of the antigen as compared with the P-Lip-L7. Antibody titre was significantly less in the animals immunized with S-19 or IFA-L7 combination. High antibody response was maintained in E-Lip-L7 immunized mice following each booster with maximum antibody level on seventh day after the last immunization as compared with P-Lip-L7 group which was unable to rise to that extent. The antibody response evoked by S-19 or IFA-L7 was significantly less as compared to E-Lip-L7 immunized group. It was observed that E-Lip-L7 maintained significantly higher titer of IgG1 and IgG2a as compared with P-Lip-L7-immunized group. Both S-19 as well as IFA-L7 combination failed to induce significant level of IgG1 and IgG2a titre in the serum after 42 days post-first immunization and the trend was maintained even after day 60 post-immunization (Mallick et al., 2007). BALB/c Four-to-six-week-old female Balb/c mice were randomly distributed into eight experimental groups so that each group consisted of 20 animals. Each group consisted of 20 animals. Various groups of mice were injected separately through subcutaneous route, on days 0,21, and 28 with escheriosome entrapped rL7/L12 (E-Lip-L7), egg PC/Chol liposome entrapped rL7/L12 protein (P-Lip-L7), rL7/L12 protein with Complete Freund’s adjuvant (IFA-L7), rL7/L12 protein alone (free form; F-L7), E. coli lipid sham liposome and sham escheriosome mixed with free rL7/L12 protein, a physical mixture (EL + L7). The positive control in this study were mice vaccinated with about 5 x 10^6 cfu of Brucella S-19. The negative control for this study received only PBS. Each animal was immunized with a priming dose of 50 micrograms of rL7/L12 protein per animal (day 0) and boosted with 30 micrograms per animal (days 21 and 28) (Mallick et al., 2007). Escherischia coli escheriosome-mediated cytosolic delivery of recombinant rL7/L12 protein can elicit strong immunological responses in the Balb/c mice. However, egg PC/Chol liposome entrapped rL7/L12 was found to impart relatively poor immune response. Escheriosome-entrapped rL7/L12 protein elicited high IgG2a isotype response, suggestive of its relevance in imparting protection against brucellosis in mice(Mallick et al., 2007). One week after the final immunization, the mice belonging to various groups were challenged with 2 x 10^5 cfu of a virulent culture of B. abortus 544 intraperitoneally in 0.2ml of saline solution. After 7, 15 and 30 days post-challenge, four mice from each group were euthanatized and their spleens were analyzed (Mallick et al., 2007). licensed Brucellosis human vaccine Generic Unknown VO_0000025 Live, attenuated vaccine Licensed A generic representation of live, attenuated vaccines historically used to prevent Brucellosis infection in humans. These vaccines contain weakened strains of Brucella bacteria to stimulate protective immunity without causing disease. Live attenuated B. abortus with deletion of znuA VO_0000386 Live, attenuated vaccine The znuA-deleted mutant is very attenuated (Yang et al., 2006). ΔznuA mutant, RB51strains were grown overnight in Brucella broth at 37°C. Cells were pelleted, washed twice in sterile phosphate-buffered saline, and diluted to 1 × 10^8 cells/200 μl in sterile PBS. The actual viable inoculum CFU was confirmed by serial dilution tests on Bacto Potato Infusion Agar. Each vaccine consisted of 0.2 ml of this suspension (Yang et al., 2006). The antigen for this vaccine is live, attenuated B. abortus strain 2308 with a znuA knockout mutation (Yang et al., 2006). Gene mutation A suicide plasmid and a B. abortus mutant were constructed, then plasmids from transformants were isolated and analyzed by agarose gel electrophoresis after restriction digestion. B. abortus ΔznuA was electroporated with pBznuA to finalize the mutation (Yang et al., 2006). BALB/c Experiments were conducted with 7- to 9-week-old age-matched mice. 0.2 ml of vaccine was administered to mice via intraperitoneal injection (Yang et al., 2006). Compared to wild-type strain 2308, splenic CFU in B. abortus ΔznuA-dosed mice were significantly decreased at weeks 1, 4, and 8. Importantly, by week 8, in three of the five B. abortus ΔznuA-dosed mice, splenic CFU could not be detected (Yang et al., 2006). The results showed that the ΔznuA mutant was mostly cleared by 8 weeks postinfection. Splenic CFU detected 4 weeks postchallenge were all wild type, and none were from the ΔznuA mutant. Protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain, so this mutant is a potential live vaccine (Yang et al., 2006). For challenge study, B. abortus 2308 was diluted in sterile PBS, in which 100 μl of bacterial suspension contained 5 × 10^4 CFU bacteria, and immunized and naive mice were subsequently challenged intraperitoneally. The challenge dose was confirmed by plating B. abortus on PIA (Yang et al., 2006). Live, Attenuated Rifampin-Resistant Rough Mutants of Brucella melitensis VO_0004147 Live, attenuated vaccine The vaccines were made through suspensions of B. melitensis mutant strains at a concentration of 5 × 10^8 CFU/ml (Adone et al., 2005). This vaccine contains rifampin-resistant rough mutants of Brucella melitensis (Adone et al., 2005). Microencapsulated B. melitensis vjbR mutant vaccine VO_0000398 Live, attenuated vaccine The vaccine was stored at 4°C (Arenas-Gamboa et al., 2008). B. melitensis mutant bjvR::Tn5 is an attenuated live strain. 6 x 10^6 CFU of the live B. melitensis mutant bjvR::Tn5 was suspended in 1ml of MOPS buffer (buffer containing MOPS and NaCl) and mixed with 5 ml of alginate solution. Spheres were obtained by extruding the suspension through a 200 micron nozzle into a 100 mM calcium chloride solution and stirred for 15 minutes. After extrusion of the bacteria-alginate mixture into the CaCl2, the capsules were washed twice with MOPS for 5 minutes and further crosslinked with 0.05% poly-L-lysine for 10 minutes. Following two successive washes, the beads were stirred for five minutes in a solution of 0.03% (w/v) alginate to apply a final outer shell and washed twice with MOPS (Arenas-Gamboa et al., 2008). An attenuated mutant of Brucella meletensis called vjbR::Tn5 (BMEII1116) is the antigen used in this vaccine (Arenas-Gamboa et al., 2008). Gene mutation Live B. melitensis attenuated mutant vjbR::Tn5 was generated by mutation of the vjbR gene through interruption with a Tn5 (Arenas-Gamboa et al., 2008). Immunization with BM vjbR::Tn5 elicited an IgG response that was clearly detectable after 2 weeks post-vaccination for either encapsulated or nonencapsulated vaccines. In mice vaccinated with the nonencapsulated BM vjbR::Tn5 vaccine, IgG levels peaked at 8 weeks post-immunization, whereas IgG responses in mice immunized with the BM vjbR::Tn5 with VpB in the shell, levels increased steadily and reached a maximum after 18 weeks post immunization. In this case, an induction of higher and sustained antibody levels correlated with enhanced protection for both BM vjbR::Tn5 in alginate and vjbR::Tn5 in VpB shell formulations. Mice vaccinated with the encapsulated vjbR::Tn5 revealed elevated secretion from spleen cells of INF-γ, IL-12, but no IL-4. These suggested an induction of a T helper 1 (Th1) response reflecting the enhanced immunity associated with microencapsulation (Arenas-Gamboa et al., 2008). BALB/c Sixty female BALB/c mice were randomly distributed into groups of 10 for intraperitoneally (IP) vaccination. Each animal was given a single dose of microcapsules containing 1 x 10^5 CFU of either: encapsulated B. melitensis mutant vjbR::Tn5 in alginate (vjbR::Tn5/alginate), encapsulated vjbR::Tn5 in alginate with VpB inside the capsule (vjbR::Tn5/VpB core), or encapsulated vjbR::Tn5 in alginate with VpB in the shell of the sphere (vjbR::Tn5/VpB shell). The control groups received 1 x 10^5 CFU of either nonencapsulated vjbR::Tn5, empty capsules (no bacteria entrapped), or two hundred microliters of MOPS buffer (Arenas-Gamboa et al., 2008). There appears to be a brief period between week one and two during which the organism replicates and the numbers of B. melitensis vjbR::Tn5 in the spleen increase, but by three weeks post-inoculation the number of organisms in the spleen exhibits a drastic decline and drops below the level of detection by four weeks post-infection (Arenas-Gamboa et al., 2008). A single immunization dose in BALB/c mice with the encapsulated vjbR mutant improved protection against wild-type B. melitensis 16M challenge compared to the nonencapsulated vaccine strain (P<0.05) . At certian times after vaccination, mice were challenged IP using 1 x 10^5 CFU/mouse of B.melitensis wild-type 16M. One week post challenge, mice were euthanizediation and their spleens were removed . Cytokine secretion from spleen cells of mice vaccinated with the encapsulated vjbR::Tn5 revealed significantly elevated secretion of gamma interferon compared to non-vaccinated mice 10 and 30 weeks post vaccination (Arenas-Gamboa et al., 2008). Cytokine secretion from spleen cells of mice vaccinated with the encapsulated vjbR::Tn5 revealed significantly elevated secretion of interleukin-12 compared to non-vaccinated mice 10 and 30 weeks post vaccination (Arenas-Gamboa et al., 2008). NPAP Brucella vaccine Brucella vaccine using nonpathogenic alphaproteobacteria VO_0000450 Inactivated or "killed" vaccine incomplete Freund's adjuvant Not noted. Bacterial cultures were grown on plates, then centrifuged at 15,000 x g. Bacterial pellets were washed with PBS, then the bacteria were heat-killed. These suspensions were centrifuged at 15,000 x g again, then washed with Tris buffer. The cells were suspended in Tris buffer , then disrupted with a French press. Bacterial cells were broken by two passages and then digested for 1 hour with D. Nase and R. Nase. Unbroken cells were seperated by centrifugation. Particulate matter was pelleted by centrifugation, and the resulting supernatent was stored for vaccine (Delpino et al., 2007a). The angtiens for this vaccine were Ochrobactrum anthropi, Sinorhizobium meliloti, Mesorhizobium loti, Agrobacterium tumefaciens, or Brucella melitensis H38. These were used because provious findings indicate that Brucella antigens and those from Nonpathogenic Alphaproteobacteria (NPAP) are cross-recognized by the immune system (Delpino et al., 2007a). Subcutaneous immunization with with heat-killed bacteria elicited a strong antobody response against CYT antigens. Antibody levels were highest after the second immunization, in most cases. In mice immunized with nonpathogenic species. Anti-Brucella antibody levels were augmented in all groups compared to preimmunization levels (Delpino et al., 2007a). BALB/c Mice were immunized with 3 doses (at 2 week intervals) of heat-killed bacteria (1 x 10^9 CFU per dose) mixed with adjuvant and administered subcutaneously in the back or with 3 doses of CYTs administered intraperitoneally. Control groups received PBS. The Brucella group was immunized with B. Melitensis H38 (Delpino et al., 2007a). Not noted. Cross-reacting anibodies were detected in serum samples obtained at the moment of challenge in all groups of mice. Spleen counts were significantly lower in immunized mice than in mice injected with PBS. Protection levels for nonpathogenic bacteria were lower than that obtained with Brucella immunization . Counts of CFU in mice immunized with B. melitensis were significantly lower than those of mice immunized with any other bacteria. Anti-brucella antibodies were detected in all groups 30 days postchallenge at levels similar to those found at the moment of challenge (Delpino et al., 2007a). One month after the final immunization, mice were challenged intravenously with 1.3 x 10^4 CFU of live B. abortus 2308. The mice were killed 30 days later and had their spleens analyzed and plated (Delpino et al., 2007) Anti-Brucella IgG levels were significatly higher than preinfection levels in all groups of mice infected with NPAP. Mice infected with HKBA exhibited a significant rise in anti-Brucella IgG in serum. No significant increase in IgA was detected at the time of challenge for mice immunized with HKBA or infected with NPAP (Delpino et al., 2007a). BALB/c Two separate experiments of oral immunization were performed. Mice were inoculated intragastrically at weekly intervals with three doses of live NPAP(1 x 10^8 CFU) or heat-killed B. abortus (HKBA) strain 2308 (1 x 10^8 CFU)or PBS by an intragastric feeding tube (Delpino et al., 2007a). Not noted. Serum levels of anti-Brucella IgA increased significantly in mice previously infected with O. anthropi and in mice orally infected with HKBA. Fecal levels of anti-Brucella IgA also increased after challenge (Delpino et al., 2007a). Twenty days after the last infecting dose, mice were challenged by the same route with live B. abortus 2308 (Delpino et al., 2007a). Porin and S-LPS Extracted from Virulent Brucella abortus 2308 VO_0000403 Subunit vaccine The porin-S-LPS was given without adjuvant or in several adjuvants: trehalose dimycolate and muramyl dipeptide; the pluronic polymer L-121 and muramyl dipeptide; or complexed with Quil A in immunostimulating complexes (Winter et al., 1988). The S-LPS was produced from smooth strain 2308 by extraction in hot phenol followed by treatment with guanidinium thiocyanate. Inocula were prepared from a freshly thawed vial which was diluted in sterile phosphate buffered saline to yield an infecting dose of 5 x 10^4 bacteria per 0.1 ml. Each dose was mixed with its appropriate adjuvant (Winter et al., 1988). The antigen for this vaccine is a complex of porin and smooth lipopolysaccharide (S-LPS) extracted from virulent Brucella abortus 2308 (Winter et al., 1988). Protective vaccines caused the formation of increased concentrations of circulating O-polysaccharide-specific antibodies, although there were individual exceptions to the quantitative associatiQn between O-polysaccharide-specific antibodies and protection. Antibodies specific for porin or R-LPS were found in negligible quantities in vaccinated mice (Winter et al., 1988). BALB/c Five week-old female mice were used. The mice were vaccinated subcutaneously. Blood samples were taken on one or more occasions during the course of some experiments. Quantitative cultures for viable B. abortus were performed on spleens at 1 and 4 weeks postinfection. Each mouse was vaccinated with 30 micrograms of porin-S-LPS (Winter et al., 1988). Vaccination with porin-S-LPS conferred significant protection against challenge infections with either B. abortus 2308 or 19 at both 1 and 4 weeks postinfection. The magnitude of protection was not significantly enhanced at either time period by the inclusion of TDM and MDP adjuvant in the vaccine. In contrast, vaccination with the same quantity of porin-R-LPS with or without adjuvant provided no significant protection against either challenge strain (Winter et al., 1988). B. abortus smooth strain 2308 of known virulence was used for challenge infections. In some experiments, vaccination or challenge was performed with vaccine strain 19. Animals were challenged 4 weeks postinfection with approximnately 5 x 10^4 CFU of live B. abortus cells (Winter et al., 1988). Recombinant B. abortus AsnC protein with CFA/IFA adjuvant VO_0004286 Subunit vaccine Research Intramuscular injection (i.m.) CFA/IFA Intramuscular injection (i.m.) Recombinant B. abortus AsnC protein (Fu et al., 2012) Recombinant protein preparation Recombinant B. abortus CobB protein with CFA/IFA adjuvant VO_0004285 Subunit vaccine Research Intramuscular injection (i.m.) CFA/IFA Intramuscular injection (i.m.) Recombinant Brucella abortus CobB (Fu et al., 2012) Recombinant protein preparation Recombinant B. abortus RB51SOD VO_0000720 Live attenuated B. abortus strain RB51SOD is a recombinant strain of B. abortus cattle vaccine strain RB51. RB51SOD overexpresses B. abortus Cu/Zn superoxide dismutase (SOD). Overexpression of this enzyme significantly increases vaccine efficacy against strain 2308 challenge (Vemulapalli et al., 2004). RB51SOD does not increase virulence compared to RB51. RB51SOD is not more virulent than RB51 (Vemulapalli et al., 2004). The gene for B. abortus Cu/Zn SOD (sodC) along with its own promoter was initially obtained from a genomic library of B. abortus strain 2308, and later subcloned into pBBR1MCS, a broad-host-range plasmid. The resulting plasmid pBBSOD was transformed into E. coli DH5a, confirmed, purified, and then electroporated into B. abortus strain RB51. Strain RB51 containing the plasmid pBBSOD was designated RB51SOD (Vemulapalli et al., 2000a). Protein overexpression B. abortus Cu/Zn SOD is over-expressed in recombinant strain RB51SOD using the broad-range plasmid pBBR1MCS (Vemulapalli et al., 2000a). BALB/c mice RB51SOD was used to vaccinate mice with a dose of ~4 x 10^8 cfu per mouse. The presence of the Cu/Zn SOD plasmid in strain RB51 does not alter its vaccine efficacy. Overexpression of SOD does not alter the attenuation characteristic of strain RB51 (Vemulapalli et al., 2000a). VO_0003068 Recombinant strain RB51SOD in mice induces better protection against virulent B. abortus infection compared to B. abortus vaccine strain RB51 (Vemulapalli et al., 2000a). None observed. Mice vaccinated with RB51SOD, but not RB51, develop antibodies and cell-mediated immune responses to Cu/Zn SOD (Vemulapalli et al., 2000a). Mice vaccinated with strain RB51SOD developed a Th1 immune response to the CuZn SOD, indicated by specific induction of serum IgG2a antibodies, but not IgG1 antibodies. IFN-gamma , but not IL -4 is secreted by CuZn SOD-stimulated splenocytes (Vemulapalli et al., 2000a). The enhanced protective immunity conferred by strain RB51SOD my be attributed to the specific cell-mediated responses, especially IFN-gamma secretion (Vemulapalli et al., 2000a). Recombinant B. abortus RB51WboA VO_0000404 Live attenuated B. abortus strain RB51WboA is not more virulent than RB51 (Vemulapalli et al., 2000b). RB51WboA is a recombinant Brucella strain that complements RB51 with a functional wboA gene. Rough Brucella abortus RB51 is a stable, attenuated mutant vaccine generated strain derived from virulent strain 2308. The wboA gene in RB51 is disrupted by an IS711 element. Abrogation of the wboA gene in smooth , virulent B. abortus, B. melitensis , and B. suis results in rough , attenuated mutants which fail to produce O polysaccharide (O antigen) (Vemulapalli et al., 2000). Groups of 13 mice each were vaccinated by intraperitoneal (i.p.) inoculation of viable strains RB51 (3 × 10^8 CFU/mouse), RB51SOD (5 × 10^8 CFU/mouse), RB51WboA (2.5 × 10^8 CFU/mouse) and RB51SOD/WboA (2.8 × 10^8 CFU/mouse) (Vemulapalli et al., 2004). No bacteria were isolated from the strain RB51WboA- and RB51SOD/WboA vaccinated mice at the time of challenge (Vemulapalli et al., 2004). Mice vaccinated with strains RB51 and RB51SOD showed significant protection in comparison to control. Mice vaccinated with strain RB51SOD showed a significantly better protection (mean reduction in CFU/spleen = 2.9 logs) than the mice vaccinated with strain RB51 (mean reduction in CFU/spleen = 1 log) (P = 0.02). No bacteria were isolated from the strain RB51WboA- and RB51SOD/WboA vaccinated mice, indicating that these mice developed a superior immunity that prevented the infection of B. abortus 2308 (Vemulapalli et al., 2004). Seven weeks after vaccination, five mice from each group were challenged i.p. with 3 × 10^4 CFU/mouse of virulent B. abortus 2308, and the remaining five mice were challenged i.p. with 2.4 × 10^4 CFU/mouse of virulent B. melitensis 16 M. Two weeks after challenge, the mice were euthanized and the number of CFUs in their spleens were determined (Vemulapalli et al., 2004). Recombinant Brucella DnaK protein vaccine VO_0000373 Subunit vaccine Two different adjuvants were used in these vaccines: Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA). CFA was administered on day 0 and IFA was administered on day 15 (Delpino et al., 2007). The open reading frame of DnaK was cloned in the Pet17b vector (Novagen, Madison, WI, USA). The specific primers for B. abortus DnaK contain with XbaI and BamHI restriction sites at the 5′ ends: sense 5′ GCAGTTTCTAGAATGGAGAGAAATA 3′, antisense 5′ TAAAAGGATCCAATTACGACGAC 3′. B. abortus genomic DNA was used as template for PCR with Pfu DNA polymerase (Stratagene). Plasmid Pet17b was digested with BamHI and NheI. After ligation, the mix was used to transform E. coli JM109 competent cells. The plasmid DNA of a clone containing the insert was purified and used to transform E. coli strain BL21 (DE3) competent cells. Upon induction with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), recombinant DnaK (rDnaK) was successfully expressed, and purified using a Mono Q column in an AKTA apparatus (Amersham Pharmacia, Uppsala, Sweden). Recombinant proteins were adsorbed with Sepharose-polymyxin B (Sigma, St. Louis, MO) to eliminate lipopolysaccharide contamination (Delpino et al., 2007). The antigen for this vaccine is rDnaK protein from B. abortus strain 2308, B. abortus strain S19, and Brucella melitensis strain H38 (Delpino et al., 2007). Immunization with rSurA elicited a vigorous IgG response that was detectable after the first immunization, increased further after the second Ag injection. PBS-immunized animals challenged with B. abortus 2308 developed antibodies against rSurA at 1 month after infection. Anti-rSurA IgG2a titers were higher than IgG1 titers during the whole immunization. rSurA stimulated significant production of IFN-γ, IL-2, IL-4 and IL-5 in spleen cells from rSurA-immunized animals but not from the PBS control group. All animals immunized with rDnaK alone elicited a humoral immune response that was detectable 15 days after the first immunization and increased further after the second injection to reach an IgG mean titer of 217,000 at day 30 post-vaccination. Immunization with rDnaK plus adjuvant induced similar anti-rDnaK IgG titers than immunization with rDnaK alone. None of the animals inoculated with PBS showed specific anti-rDnaK Abs at the time of challenge but notably, 30 days after infection all of them produced anti-rDnaK. Stimulation with rDnaK induced a significant production of IFN-γ and IL-2 in spleen cells from all mice immunized with rDnaK plus adjuvant Cells from rDnaK alone- or PBS-immunized mice were unable to stimulate the secretion of IFN-γ, IL-2, IL-4, IL-5 or IL-10 in response to rDnaK (Delpino et al., 2007b). BALB/c Female BALB/c mice of 6 to 8-weeks old were used for the testing of these vaccines. Mice were anaesthetized with methoxyfuorane and immunized by the intraperitoneal route with (i) 30 μg of rSurA, (ii) 30 μg of rDnaK, (iii) rSurA + rDnaK (30 μg + 30 μg) or iv) PBS, which was the negative control. Antigens and PBS were administered mixed with Complete Freund's Adjuvant (CFA) on day 0 and with Incomplete Freund's Adjuvant (IFA) on day 15. A fifth group of mice was immunized i.p with 30 μg rDnaK in PBS without no adjuvant on days 0 and 15. As reference vaccinated controls other groups were immunized once (i) by the subcutaneous route at day 0 with 8 × 10^8 formalin-killed B. melitensis H38S in IFA or (ii) i.p with 1 × 10^4 live B. abortus S19. Two separate assays of immunization were performed. The first experiment included groups immunized with rDnaK plus adjuvant, rDnaK without adjuvant, rSurA, and the negative (PBS) and reference (H38) control groups. The second experiment included groups immunized with rDnaK, rSurA, and rDnaK + rSurA, all with adjuvant, and the negative (PBS) and reference (B. abortus S19) control groups. Sera for antibody detection were obtained by retro-orbital bleeding under anaesthesia at 15, 30, 45 and 75 days after the first immunization (Delpino et al., 2007b). Mice given rSurA or rDnaK plus adjuvant exhibited a significant degree of protection against B. abortus when compared with controls receiving PBS. Formalin-killed B. melitensis H38S, the control vaccine, induced 2.19 units of protection against B. abortus. Immunization with rDnaK alone induced a low but still significant level of protection. In a second experiment, both evaluated vaccines (rDnaK or rSurA plus adjuvant) induced significant protection against B. abortus infection. There was no additive protection by the simultaneous immunization with both rDnaK and rSurA. All evaluated vaccines induced less protection than H38 or B. abortus strain 19 control vaccines. Altogether these results indicate that rSurA or rDnaK in adjuvant induce partial protection against B. abortus infection (Delpino et al., 2007b). Immunized mice were challenged by i.p. injection with 1 × 104 B. abortus 2308 (Delpino et al., 2007b). Recombinant chimera vaccine BLSOmp31 VO_0000413 Subunit vaccine The chimerical protein BLSOmp31 is synthesized using parts of Brucella Lumazine Synthase (BLS) and an outer membrane protein of 31 kDa (Omp31), which have given limited protection in mouse models (Cassataro et al., 2007). None. Recombinant BLS (rBLS), rOmp31 and rBLSOmp31 were expressed in E. coli and purified as previously described (Laplagne et al., 2004). Purity was assessed by Coomassie blue stain, and recombinant proteins were adsorbed with Sepharose-polymyxin B to eliminate LPS contamination. The peptide was further purified by HPLC using a C-18 reverse phase column and the molecular weight was confirmed by mass spectroscopy (Laplagne et al., 2004). The antigen used in this vaccine is a chimerical protein containing parts of Brucella Lumazine Synthase (BLS) and an outer membrane protein of 31 kDa (Omp31). Immunization with recombinant BLSOmp31 elicited a strong specific IgG response that was detectable after the first immunization, increased after the second boost and reached IgG mean titers of 78,400 or 26,000 (anti-rOmp31 and anti-Omp31(48–74) , respectively) at the time of bacterial challenge. Immunizatio with rBLSOmp31 elicited high levels of anti-Omp31 IgG1 as well as IgG2a antibodies (IgG1 mean titer: 17,600; IgG2a mean titer: 5300). IgG1 titers predominated over IgG2a titers during the whole immunization schedule for both recombinant antigens. The elicited anti-peptide antibodies recognized the recombinant Omp31 as well as the native membrane protein, as demonstrated by the reactivity of the sera against whole rough B. ovis bacteria. These antibodies also produced complement mediated B. ovis cells lysis (Cassataro et al., 2007). Mice (eight/group) were anaesthetized with methoxyfuorane and immunized by the intraperitoneal route with 30 The chimera significantly increases the protection elicited against B. ovis with respect to either BLS or Omp31. In fact, rBLSOmp31 induced the highest protection level (2.45 log) against B. ovis, which was only comparable with that induced by the control vaccine (2.42 log), but significantly higher (P < 0.01) than the vaccination with rBLS plus Omp31(48–74) (1.08 log) (Cassataro et al., 2007). Immunized mice were challenged, by intravenous injection, with 1×10^4 B. melitensis H38S or 1×10^4 B. ovis. Mice were killed by cervical dislocation 30 days after being challenged and their spleens were removed aseptically. Each spleen was homogenized in a stomacher bag, serially diluted, plated on supplemented TSA yeast extract (TSA-YE) and incubated (Cassataro et al., 2007). Recombinant O. anthropi 49237SOD VO_0000407 Recombinant vector vaccine Ochrobactrum anthropi is very closely related to Brucella, and has been used as a vaccine or vaccine vector for Brucellosis (He et al., 2002). O. anthropi strains are rarely pathogenic to humans (He et al., 2002) Ochrobactrum anthropi strain 47237 was originally isolated from soil. Plasmids pBBSOD and pBBR1MCS were electroporated into electrocompetent O. anthropi strain 49237 using a protocol described previously for Brucella. Colonies of the O. anthropi strain 49237 containing pBBSOD or pBBR1MCS (designated O. anthropi strain 49237SOD or 49237pBB, respectively) were selected from TSA plates containing chloramphenicol at a concentration of 30 μg/ml (He et al., 2002) Experiment 1 (Exp. 1): Groups of eight mice were inoculated (i.p.) with either saline (negative control), B. abortus strain RB51 (2×10^8 CFU/mouse; positive control), recombinant B. abortus strain RB51SOD (2×108 CFU/mouse; positive control), O. anthropi 49237 (5×10^8 CFU/mouse), O. anthropi 49237pBB (5 × 108 CFU/mouse), or O. anthropi 49237SOD (5×10^8 CFU/mouse). At 2, 4, and 6 weeks after inoculation, three mice from each group were bled retroorbitally. The sera obtained were stored at −40°C until use in an enzyme-linked immunosorbent assay (ELISA) or for Western blot analysis. In a second experiment (Exp. 2) whether multiple injections were required for protection was asessed . Groups of five mice were immunized i.p. with either saline, B. abortus strain RB51, or O. anthropi 49237SOD at the doses described above. After two weeks, mice inoculated with O. anthropi 49237SOD were reimmunized with O. anthropi 49237/SOD (5×10^8 CFU/mouse). To determine whether different doses influenced the results of protection, four groups of five mice each were inoculated with O. anthropi 49237 at four different doses: 5×10^8, 5×10^7, 5×10^6, and 5×10^5 CFU/mouse. A fifth group (five mice) was inoculated i.p. with saline as negative control. Synthetic CpG-containing oligodeoxynucleotides (CpG-ODN) were administered to group four as an immunostimulatory adjuvant. One group (eight mice) was inoculated i.p. with O. anthropi strain 49237 (5×10^8 CFU/mouse); one group with strain 49237 (5×10^8 CFU/mouse) and CpG adjuvant, one group with strain 49237SOD alone (5×10^8 CFU/mouse), and one group with strain 49237SOD (5×10^8 CFU/mouse) and CpG adjuvant. The CpG adjuvant (10 nmol) was administered i.p. 4 h before inoculation and again at the time of inoculation of the bacterial strains. Three groups of mice (eight/group) served as controls and were inoculated with saline alone, CpG-ODN alone, or E. coli DH5α (10^6 CFU/mouse) and CpG-ODN. The mice were bled at 2, 4, and 6 weeks after inoculation (He et al., 2002). Six weeks after inoculation, mice (5) from each group were challenged i.p. with 2×10^4 CFU of virulent B. abortus 2308/mouse. The mice were sacrificed after two weeks. Their spleens were homogenized, and spleens from the dilutions were plated to determine the numbers of Brucella CFU per spleen. The remaining unchallenged mice (each group) were sacrificed 6 to 8 weeks postinoculation, and spleen cells collected for cell culture in vitro (He et al., 2002). AsnC Brucella abortus 2308 3788213 489054600 BAB_RS24000 JMRZ01000003 WP_002964767 359391 I 1633929 1634399 - ArsR family transcriptional regulator 5.32 15857.78 156 >NC_007618.1:1633929-1634399 Brucella melitensis biovar Abortus 2308 chromosome I, complete sequence, strain 2308 TCTAACGCCCATCCAGCGGGACGGCTGTGGTGTCCTTGATCTCTTCCATCACGAAAGAGGCCGATACATC CGATAGCGGTACACAGGCTATGAGGCGCTGGTAGAGCCGGTCATAAGCTCTGACATCGGAAATGCGTGCC TTCAGCACATAGTCCAGATCGCCGGTCATGCGATAGGCGCCGACGATTTCCGGGAAGCGAGCCACGGCAG CGCGGAATTTGTTCAGCCATTCGGGATCATGGGCGGAGGTGCGCACCAGAATGAAAACCGAGAGGCCATA GCCGACCATTTCCGCATCCACCAGCGCCACGCGACCTTTGATGACGCCATCGTCTTCCATGCGCTTTACG CGCCGCCAGCAGGCATTGCGCGAAAGATTCACCCGTTCGGAAAGGGAATCAACCGACAAAGTGCCGTCGT TTTGCAACTCAGCCAGGATTTTTCGGTCTATATCGTCCATAGATGCTGTCA >WP_002964767.1 MULTISPECIES: Lrp/AsnC family transcriptional regulator [Brucella] MTASMDDIDRKILAELQNDGTLSVDSLSERVNLSRNACWRRVKRMEDDGVIKGRVALVDAEMVGYGLSVF ILVRTSAHDPEWLNKFRAAVARFPEIVGAYRMTGDLDYVLKARISDVRAYDRLYQRLIACVPLSDVSASF VMEEIKDTTAVPLDGR Protective antigen Mice given BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19 [Ref2099:Fu et al., 2012]. asp24 2352999 2353000 AAB69346.1 CDD:151121 235 ? acid shock protein 184 Secreted protein acidic and rich in cysteine Ca binding region; pfam10591 >gi|2353000|gb|AAB69346.1| acid shock protein [Brucella abortus] MESRTMKSKTAFIAVIATSLFAIPALAQEKSNANTDKPAAEAAVSSNQDAKNKRGPIDLEKFSRMDQLKA ADTNGDGVLSRDEIEALALKRVVSRAADRMERRLDVNGDGKVTLDEIQNQRKKEFAALDRNDDGKLDRHE MRAAKMSHRGHKGHHEMGKHHKSGDHKGGDHKGPMKTKAQKPQE Virmugen A asp24 mutation in Brucella abortus is attenuated and provides significant protection in mice [Ref694:Kahl-McDonagh et al., 2007]. B. abortus strain 19 L7/L12 Strain 19 VO_0010928 304271 L19101 CDD:234671 CDD:100102 235 ? ribosomal protein L7/L12 4.49 11795.56 183 biovar Abortus >gi|304270|gb|L19101.1|BRURPL712X Brucella abortus ribosomal protein L7/L12 gene, complete cds CAAGCTCGTTATTCTCGGTGGCGCAATGGGTGCAACGACGCTTAGACCGATGGCGTCAAGTCGCTTCGTT CGCTCCCGTCGCTCGACGAACTGCGCGCAAAGCTGGTTGGTATGATCCAGACCCCGGCTCAGCGTCTTGC AGTGCTTACCAGCGCTCCGCGGGCCAGATCGCCCGCGTTATTGCGCGCACGCCCGGAAGAACGAGGCGGC TTAAGGCCGTTTCTCGCTGTCAACAGTTCAAACCTTAATTATAGGAAATACAAAAATGGCTGATCTCGCA AAGATCGTTGAAGACCTTTCGGCCCTGACCGTTCTGGAAGCCGCTGAGCTGTCCAAGCTTCTCGAAGAGA AGTGGGGCGTTTCGGCTGCTGCTCCGGTCGCTGTTGCTGCTGCCGGTGGCGCTGCCCCTGCTGCTGCCGC AGAAGAAAAGACCGAATTCGACGTCGTTCTCGCTGACGGCGGCGCTAACAAGATCAACGTGATCAAGGAA GTGCGCGCACTCACCGGTCTCGGCCTCAAGGAAGCCAAGGACCTGGTCGAAGGCGCTCCGAAGGCTGTCA AGGAAGGCGCCTCGAAGGACGAAGCTGAGAAGATCAAGGCACAGCTCGAAGCTGCTGGCGCCAAGGTTGA ACTCAAGTAAGTTTGGACTATT >AAA19863.1 ribosomal protein L7/L12 [Brucella abortus] MADLAKIVEDLSALTVLEAAELSKLLEEKWGVSAAAPVAVAAAGGAAPAAAAEEKTEFDVVLADGGANKI NVIKEVRALTGLGLKEAKDLVEGAPKAVKEGASKDEAEKIKAQLEAAGAKVELK Protective antigen B. melitensis M5 P39 B. melitensis strain M5 VO_0010948 122892474 EF189139 ABM67295 CDD:224567 CDD:304360 1004957 ? immunogenic 39-kDa protein 4.69 40682.69 483 biovar: 1 str. M5 >gi|122892473|gb|EF189139.1| Brucella melitensis strain M5 immunogenic 39-kDa protein gene, complete cds ATGGGTACGGCTTCCTGTGGTTTGCTCGCCGGAATGGCGCCTGTTGCCAATGCGCAGGAAAAGCAGAATG TCGAGGTTCTGCACTGGTGGACGTCCGGCGGCGAAGCGTCCGCGCTTGAAGTTTTGAAAAAAGATCTTGA AAGCAAGGGCATTAGCTGGACCGATATGCCGGTTGCAGGTGGCGGCGGCACGGAAGCCATGACCGTTTTG CGCGCGCGCGTTACCGCAGGCAATGCGCCAACCGCGGTGCAGATGCTGGGTTTTGACATTCGCGACTGGG CGGAGCAGGGCGCACTCGGCAATCTCGATACGGTTGCTTCCAAGGAAGGCTGGGAAAAGGTTATTCCGGC TCCCTTGCAGGAATTTGCCAAATATGACGGCCACTGGATTGCTGCGCCCGTCAATATTCACTCCACCAAC TGGATGTGGATCAACAAGGCTGCTCTCGACAAGGCTGGCGGCAAGGAGCCGACCAATTGGGATGAGCTGA TTGCGCTTCTCGACAATTTCAAGGCGCAGGGCATTACGCCGATCGCGCATGGCGGCCAGCCGTGGCAGGA TGCAACCATTTTCGATGCGGTTGTTCTTTCATTCGGCCCGGATTTCTACAAGAAGGCCTTCATCGATCTC GACCCGGAAGCACTGGGCAGCGATACCATGAAGCAGGCCTTCGACCGCATGTCCAAGCTTCGCACCTATG TTGATGACAACTTCTCCGGCCGTGACTGGAACCTTGCTTCGGCCATGGTTATCGAAGGCAAGGCCGGTGT CCAGTTCATGGGCGACTGGGCGAAGGGCGAGTTCCTCAAGGCGGGCAAGAAGCCGGGTGAGGATTTCGTC TGCATGCGTTATCCGGGCACGCAGGGTGCTGTCACTTTCAATTCCGGCATGTTCGCCATGTTCAAGGTTT CGGAAGACAAGGTTCCCGCACAGCTTGAAATGGCTTCGGCGATTGAAAGCCCTGCCTTCCAGTCTGCCTT TAATGTGGAGAAGGGGTCGGCCCCGGCACGCACGGATGTGCCCGATACCGCTTTCGATGCCTGTGGCAAG AAGACCATTGCCGATGTCAAGGAAGCAAACAGCAAGGGCACTCTGCTTGGCTCCATGGCGCATGGCTATG CCAATCCGGCTGCCGTGAAGAATGCGATCTACGACGTCGTGACCCGCCAGTTCAACGGCCAGCTTTCTTC GGAAGATGCCGTCAAGGAACTCGTTGTGGCGGTTGAAGCCGCAAAATAAGGCCACGCGGAAACGTCCGGG GGAACCTCCCGGACGTTTTTCCTGCGTGCTCCAGCAGAAGCGCATAGGCGGTTGGCGCAGCTAACCGGCA CTTACGACGGAATTGCATGCCGATATGCCCGCCTCACCATCCGAGGCGCGTTTCGTGGGACATAGAGC >ABM67295.1 immunogenic 39-kDa protein [Brucella melitensis bv. 1 str. M5] MAPVANAQEKQNVEVLHWWTSGGEASALEVLKKDLESKGISWTDMPVAGGGGTEAMTVLRARVTAGNAPT AVQMLGFDIRDWAEQGALGNLDTVASKEGWEKVIPAPLQEFAKYDGHWIAAPVNIHSTNWMWINKAALDK AGGKEPTNWDELIALLDNFKAQGITPIAHGGQPWQDATIFDAVVLSFGPDFYKKAFIDLDPEALGSDTMK QAFDRMSKLRTYVDDNFSGRDWNLASAMVIEGKAGVQFMGDWAKGEFLKAGKKPGEDFVCMRYPGTQGAV TFNSGMFAMFKVSEDKVPAQLEMASAIESPAFQSAFNVEKGSAPARTDVPDTAFDACGKKTIADVKEANS KGTLLGSMAHGYANPAAVKNAIYDVVTRQFNGQLSSEDAVKELVVAVEAAK Protective antigen The 39-kDa protein is immunogenic. BAB1_0278 Brucella melitensis biovar Abortus 2308 82615279 CDD:264424 EnsemblGenomes-Gn:BAB1_0278 EnsemblGenomes-Tr:CAJ10234 InterPro:IPR011681 UniProtKB/TrEMBL:Q2YPC5 359391 ? conserved hypothetical protein 8.51 8787.28 148 GcrA cell cycle regulator; cl11564 >CAJ10234.1 conserved hypothetical protein [Brucella abortus 2308] MHWKSRSPLSEALPMFDKLTSPHEGFRLADLSRRQCKWPVNRAQAGELHLFCGEAVQNGHPYCEEHCGKA YTGKAGSR Protective antigen BCSP31 Brucella abortus 144104 144105 AAA22993.1 CDD:273982 235 ? cell surface protein 7.77 32204.04 397 biovar Abortus >gi|144104|gb|M20404.1|BRUBCSP B.abortus BCSP31 gene encoding a 31-KDa cell surface protein, complete cds GCGCCTATACGCGCGACCTGATCCAGTCTTTTGCATCGCGGTGCCATGTGCGTCTGGTGGGGGCCGACGG GCTGGCGGCGATTGCCGAAGCGCATATTCGCGGTGAAAGTTTCGATGAGGCGCTGGTCATGGCGCAGATC GCGCCATGTTTTATCGAAAAGGATGGCAAGCGCACCGATATCGTGGTGCTTGCCTGTACGCATTATCCGT TTCTCGTCAATGTATTGCGCCGTCTGGCTCCATGGCCGGTGGATTGGCTGGACCCGGCGGAAGCCATTGC ACGACGCATGAAATCGCTTTTGCCTGCGCGAAGTGACGATGATGAATTTCATTCTCAAGATGACCTAGCA TTCTTCACATCCAGGAAACCCGACTATGCCATTCGCCGCCTGATGCAGGGTTTTGGGCTGCGTTTTTAAT CGTTTCAGTCGGCTCTGGCGGCGCTTGTCGGCAAGCGATTGTATTCTTTGGGAAAATCCAGAATAATGGA ATGCGGTGGTTGACAATCGGCCTCAAGCTTCCTATGGTTTTCGGCATAATCTATGCGGGAAGAGGACTGG TATTATGAAATTCGGAAGCAAAATCCGTCGCTTGGCTGTTGCGGCGGTGGCGGGCGCGATTGCGTTGGGA GCGAGCTTTGCGGTTGCACAGGCCCCGACATTTTTCCGTATCGGCACTGGCGGCACAGCCGGAACCTATT ATCCGATTGGTGGTCTGATCGCGAACGCGATTTCCGGCGCAGGCGAAAAGGGCGTGCCGGGTCTCGTCGC GACGGCCGTTTCGTCGAATGGCTCGGTTGCCAATATCAATGCGATCAAGTCGGGCGCTCTGGAGTCCGGC TTTACGCAGTCAGACGTTGCCTATTGGGCCTATAACGGCACCGGCCTTTATGATGGCAAGGGCAAGGTGG AAGATTTGCGCCTTCTGGCGACGCTTTACCCGGAAACGATCCATATCGTTGCGCGTAAGGATGCAAACAT CAAATCGGTCGCAGACCTGAAAGGCAAGCGCGTTTCGCTGGATGAGCCGGGTTCTGGCACCATCGTCGAT GCGCGTATCGTTCTTGAAGCCTACGGCCTCACGGAAGACGATATCAAGGCTGAACACCTGAAGCCGGGAC CGGCAGGCGAGAGGCTGAAAGATGGTGCGCTGGACGCCTATTTCTTTGTGGGCGGCTATCCGACGGGCGC AATCTCGGAACTGGCCATCTCGAACGGTATTTCGCTCGTTCCGATCTCCGGGCCGGAAGCGGACAAGATT CTGGAGAAATATTCCTTCTTCTCGAAGGATGTGGTTCCTGCCGGAGCCTATAAGGACGTGGCGGAAACAC CGACCCTTGCCGTTGCCGCACAGTGGGTGACGAGCGCCAAGCAGCCGGACGACCTCATCTATAACATCAC CAAGGTTCTCTGGAACGAGGATACACGCAAGGCACTCGATGCGGGCCATGCGAAGGGCAAGCTCATCAAG CTCGATAGTGCGACGAGCAGCCTCGGTATTCCGCTGCATCCCGGCGCAGAACGCTTTTACAAGGAAGCGG GCGTGCTGAAATAATCCCTCAATGATCGGTTCCTGATATCTTATTCCGAATTGAAGGGTGACATTGCGGC AGCTCGTTATGCGCGCTGCTGCGCTCCCGTTTTCCAGAGCGGTTCCGGTTAGAACGGAATCGTTGGAACC GCTCTATCTCTTTGTTTTTACGCATTATCCGACGCAAAACCGTTTCACGCTTTTGCTGGAAATGCTCTAG CCTATTGAAATGCACGACCGGCAAAGTGGAGTTGGCCCGCATGACAGAAGAACAAAATGCAAAGCTT >AAA22993.1 cell surface protein precursor [Brucella abortus] MKFGSKIRRLAVAAVAGAIALGASFAVAQAPTFFRIGTGGTAGTYYPIGGLIANAISGAGEKGVPGLVAT AVSSNGSVANINAIKSGALESGFTQSDVAYWAYNGTGLYDGKGKVEDLRLLATLYPETIHIVARKDANIK SVADLKGKRVSLDEPGSGTIVDARIVLEAYGLTEDDIKAEHLKPGPAGERLKDGALDAYFFVGGYPTGAI SELAISNGISLVPISGPEADKILEKYSFFSKDVVPAGAYKDVAETPTLAVAAQWVTSAKQPDDLIYNITK VLWNEDTRKALDAGHAKGKLIKLDSATSSLGIPLHPGAERFYKEAGVLK Protective antigen bp26 Brucella melitensis bv. 1 str. Rev.1 265991532 CDD:194028 520464 ? outer membrane protein Bp26 250 Protein of unknown function (DUF541); cl01077 >gi|265991532|ref|ZP_06104089.1| outer membrane protein Bp26 [Brucella melitensis bv. 1 str. Rev.1] MNTRASNFLAASFSTIMLVGAFSLPAFAQENQMTTQPARIAVTGEGMMTASPDMAILNLSVLRQAKTARE AMTANNEAMTKVLDAMKKAGIEDRDLQTGGINIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELANV GKILDESVTLGVNQGGDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPPMP MPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIK Virmugen A bp26 mutant in Brucella melitensis was attenuated and provided protection against challenge in mice [Ref1988:Cloeckaert et al., 2004]. cobB Brucella abortus 2308 3787893 489054238 BAB_RS22225 JMRZ01000005 WP_002964414 359391 I 1271743 1273053 - hydrogenobyrinate a,c-diamide synthase 6.89 44156.43 436 responsible for the amidation of carboxylic groups at position A and C of cobyrinic acid or hydrogenobrynic acid >NC_007618.1:1271743-1273053 Brucella melitensis biovar Abortus 2308 chromosome I, complete sequence, strain 2308 CTCACGCCGCTTCTCCGCTAAAATCAATGACATGCATGAAGGAGCCGGAGACGGAACCAACCCGCAAGCC CGCTTCGCCAAGGTTCTCGCCCGATGCATCGCGCACCCGGAACAGGCGGTCGGCCTTTTCTTCCCGCACG ATGCTTGCATAATGGAATTCATGCGCTTTCAAAGGCATGTCCCACGGCAATCCGCCAAGCGGCTCCAGCA GGCGATAGCCAAGATGCAGTTTCCGCCTGGCAAAGCTCGTTTCCAATGGCAAAAGCCCCAGCATGGGATG GTGAACGCCTGCCGCATCCTCCAGTGTTTTCCCGAGGACCATATAGCCGCCGCACTCGCCATAGACCGTG ACGCCACGCGCGGCAGCGTCCCCTATCGCCGTGCGGAAACGGGATGCACCGGCCAATCGTTGCGCATGAA GTTCTGGATAACCACCGGGAAGATAGATTGCATCCGCATCTGCCTTCGGTGCTTCATCTGCCAGCGGGGA AAAGAAAGAGATTTCCGCACCCCGCCTGCGCCAGCCTTCAAAAAGATGCATATAGGCAAAAGCAAAGGCA TCGTCCCGCGCCACGGCAATGCGGTTTCCAAGCGGTTTCAGACGCGCGACATTGGCCATGGCGTCATAGC GCTTCGGGCGTAGCCAGATGGTCTGAAGCGCATCCATGTCGATATGCGCTTCCATCACATCGGCCGCTTG TTCGAGAAAGCTTTCAAGACCGGCATGTTCGTCCGCCTGCACCAGCCCCAGATGCCTTTCCGGCAAGGAA AGTGCCGGATCTCGCGGCAGCGCGCCCAGAACCGGGACGCCAAGGGGTGCCAGAGCGCCCCGCAGCATGG CTTCATGGCGGGGACTTCCGACACGATTGAGAATGACACCCTCAATCAGCACATCCTTGCGAAACTGGCT GAAACCCCACACCAGCGCCGCGATGGAGTGGGATTGCCGGGCGCAATCCACCACCAGCACGACTGGCAAA TCCAGAAGCCGCGCCAGATCGGCAGAAGAGCCCTTACCGTCAATCGCCCCGTCGAAAAGCCCCATCATGC CTTCGGCGACAAGAACACGCGCGCCGCTTTCCGTCATGCGGGAGGAAAGCGCACTGATGAGTTCCGGTCG CATGGCCCAGGGGTCGAGATTGAAACAATCCACGCCACTCGCCGCCCTGTGATAGGCGGGGTCGATATAA TCCGGCCCGGCCTTCACCGGCGCAAGCACCTCGCCCCTGCGTTTCAGGGCGCGCAACAAACCCAAGGTCA CGGTCGTCTTGCCCGACCCGGAGGCCGGTGCGGCAATCATGAATCCCTTCA >WP_002964414.1 MULTISPECIES: hydrogenobyrinate a,c-diamide synthase [Brucella] MKGFMIAAPASGSGKTTVTLGLLRALKRRGEVLAPVKAGPDYIDPAYHRAASGVDCFNLDPWAMRPELIS ALSSRMTESGARVLVAEGMMGLFDGAIDGKGSSADLARLLDLPVVLVVDCARQSHSIAALVWGFSQFRKD VLIEGVILNRVGSPRHEAMLRGALAPLGVPVLGALPRDPALSLPERHLGLVQADEHAGLESFLEQAADVM EAHIDMDALQTIWLRPKRYDAMANVARLKPLGNRIAVARDDAFAFAYMHLFEGWRRRGAEISFFSPLADE APKADADAIYLPGGYPELHAQRLAGASRFRTAIGDAAARGVTVYGECGGYMVLGKTLEDAAGVHHPMLGL LPLETSFARRKLHLGYRLLEPLGGLPWDMPLKAHEFHYASIVREEKADRLFRVRDASGENLGEAGLRVGS VSGSFMHVIDFSGEAA Protective antigen Mice given BAB1_1316 (CobB) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19 [Ref2099:Fu et al., 2012]. exsA Brucella melitensis biovar Abortus 2308 3787220 82699354 BAB1_0468 AM040264 YP_413928 359391 I 459774 461660 + ABC transporter ATPase 7.04 65025 628 identified by match to protein family HMM PF00005; match to protein family HMM PF00664 >gi|82698932:459774-461660 Brucella melitensis biovar Abortus 2308 chromosome chromosome I, complete sequence GATGAATGCCCCCAAGACGGTTTCTGCCGAATCCGGCGAGACGCTGAAAACATTGCGAAATCTGTGGCCC TACATGTGGCCGTCCGACAGGCCGGACCTGCGAATGCGCGTGGTCTGGGCCACATTCTATCTGGTTCTGT CCAAGATTGTGCTGATCCTCGTACCCTATTTCTTCAAATGGGTGACCAATGCGCTGAACGGCCAGCTTCA TGCGCCGGATTATATTCCGGTCGTTCTGGTCGGCGCGGTCATGCTGGTGCTGGCCTATAATGCGGCGAAA ATCGTGCAGGCTGGCCTCAACCAGCTTCGTGACGCACTTTTTGCCAGTGTTGGCCAATATGCGGTGCGCC AGCTTGCCTACAAGACCTTTGTGCATATGCATGAGCTTTCGCTGCGCTTTCATCTGGAGCGGCGCACGGG CGGCCTGTCGCGCGTCATCGAGCGCGGCACCAAGGGCATTGAAACCATCGTCCGTTTTACCATCCTGAAC ACGCTGCCTACCATTCTGGAATTTGCGCTGACGGCGGTGATCTTCGCTTTCGCCTATGGCCTGTCCTATC TCGTGGTCGTGGCGGCGACGGTCTGGCTCTATACGTGGTTCACCATCAGGGCGAGCGATTGGCGCATCAA TATCCGTCGCGAGATGAACGATTCCGATACCGACGCCAATACCAAGGCCATCGATTCGCTTCTCAACTTC GAGACGGTCAAATATTTCGGCAATGAAGCGATGGAGGCCAAGCGTTTCGATGGGGCTATGGCGCGATATG AAAAGGCTGCAACCCAGACCTGGACCTCGCTTGGCTGGCTGAACTTCGGTCAGGCCGTGATTTTCGGCGC GGGCATGGCCATTGTCATGGTCATGTCGGCCATGGAGGTGCAGAAGGGCACACAGTCGCTGGGCGATTTC GTCTTCATCAATGCGCTTTTGATGCAGCTTTCCATCCCGCTCAATTTCATCGGCTTTATCTATCGTGAAA TCCGTCAGGGCCTCACCGATATCGAGCAGATGTTCGACCTTCTGGACGTCAAGCAGGAAGTGAAAGACAA GCCCGGCGCTCCTGCGCTGAAAGTCGATAGCGGCGCAATCTCCTTCAAGGATGTGCACTTTGCCTATGAT CCGCAGCGCCCCATCCTGCGCGGCATCAGCTTCGACGTTCCCGCAGGAAAGACGGTGGCGATTGTCGGCC CTTCGGGGGCTGGTAAATCCACCATATCGCGGCTTCTGTTCCGCTTTTACGATATCCAGTCCGGTTCGAT CACCATCGATGGGCAGGATGTGCGCGACGTGACCCAGGAAAGCCTGCGCAAGTTTATCGGCATGGTGCCG CAGGATACGGTCCTGTTCAACGACACCATCGCCTATAATATCCGCTATGGCCGCACCGATGCGAGCGAGG AGGATGTGGAAAAAGCAGCCGAGCTCGCGCAGATTGCCGGCTTCATCAAGCATCTGCCGGATGGCTACAA ATCCATGGTTGGCGAGCGCGGCCTGAAGCTTTCCGGCGGCGAAAAGCAGCGTGTGGCGATTGCCCGCACG ATCCTCAAGGCCCCGCCTATTCTTATTCTGGACGAGGCGACCTCGGCACTCGATACCGCGACCGAGCAGG ACATCCAGTCGGCGCTTGATATTGTCAGCCGCGGGCGCACCACGCTGGTGATCGCGCATCGCCTTTCCAC CGTGATCGGTGCGGATGAGATCATCGTCCTGAAAGACGGCCTGATCGCCGAGTGCGGCACGCACCGCCAT CTCCTTGACCAGAAGGGGCTTTATGCTTCCATGTGGGATCGCCAGCGCGAGGCGAGCGAGGCTGAGGAGC GCCTGCGCCAGGTGCGTGAAAGCGACGATATGGGCGTGGTTCTGCGCGGGACGCCGGCGGCGGAATA >gi|82699354|ref|YP_413928.1| ABC transporter ATPase [Brucella melitensis biovar Abortus 2308] MNAPKTVSAESGETLKTLRNLWPYMWPSDRPDLRMRVVWATFYLVLSKIVLILVPYFFKWVTNALNGQLH APDYIPVVLVGAVMLVLAYNAAKIVQAGLNQLRDALFASVGQYAVRQLAYKTFVHMHELSLRFHLERRTG GLSRVIERGTKGIETIVRFTILNTLPTILEFALTAVIFAFAYGLSYLVVVAATVWLYTWFTIRASDWRIN IRREMNDSDTDANTKAIDSLLNFETVKYFGNEAMEAKRFDGAMARYEKAATQTWTSLGWLNFGQAVIFGA GMAIVMVMSAMEVQKGTQSLGDFVFINALLMQLSIPLNFIGFIYREIRQGLTDIEQMFDLLDVKQEVKDK PGAPALKVDSGAISFKDVHFAYDPQRPILRGISFDVPAGKTVAIVGPSGAGKSTISRLLFRFYDIQSGSI TIDGQDVRDVTQESLRKFIGMVPQDTVLFNDTIAYNIRYGRTDASEEDVEKAAELAQIAGFIKHLPDGYK SMVGERGLKLSGGEKQRVAIARTILKAPPILILDEATSALDTATEQDIQSALDIVSRGRTTLVIAHRLST VIGADEIIVLKDGLIAECGTHRHLLDQKGLYASMWDRQREASEAEERLRQVRESDDMGVVLRGTPAAE Virmugen Brucella DeltaexsA mutant showed decreased survival in mice compared to the survival of parental strain S2308. This demonstrated that ExsA is critical for full bacterial virulence. The B. abortus exsA deletion mutant was used as a live vaccine candidate. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51 [Ref1786:Rosinha et al., 2002a]. IalB Brucella melitensis bv. 1 str. 16M VO_0010967 1197295 17987867 BMEI1584 AE008917 NP_540501 224914 I 1632893 1633414 + invasion protein B 8.98 17016.04 173 >NC_003317.1:1632893-1633414 Brucella melitensis bv. 1 str. 16M chromosome I, complete sequence TATGAAAAATTATCGTGCAATCGGTCTTGCATTCACGTTCACTGCCCTTTCCAGTCTTTCGGCCTTTGCA GCCTCCCTGCCCGGCGGAGCAAGCACCTTGCAGGAAACCTATCAGGACTGGACCGTGTCTTGCCAGTCGC AGAAGGATACAACAGCCTGCGTGATGCGTCAGGAGCAAAGCAGCGCCCAGGCCGGCCAGCGCGTTCTGAC TGCCGAGCTGCGCAACGTCGCCGGCGGCAAAGTTGACGGTGTGTTGCTGATGCCGTTCGGTCTTGATCTT GCCAAGGGCGCCTCGCTCAAGATTGATGACACCGCTGGGCCAAACCTCACCTTCTCCACCTGCCTGCCGC AGGGCTGCCTCGCGCCAGTGAGCTTCGATGCCAAGCAGGTTGCTGCGCTGAAATCCGGCACCAACATCAA TGTCACCACGACGGCGCTCAGCCCGAGCCAGCCGGTTGCCTTCAAGATTTCCCTGAAAGGCTTCGGTGCC GCGCTCGACCGCATTCAGGCATTGACCAAGTA >NP_540501.1 invasion protein B [Brucella melitensis bv. 1 str. 16M] MKNYRAIGLAFTFTALSSLSAFAASLPGGASTLQETYQDWTVSCQSQKDTTACVMRQEQSSAQAGQRVLT AELRNVAGGKVDGVLLMPFGLDLAKGASLKIDDTAGPNLTFSTCLPQGCLAPVSFDAKQVAALKSGTNIN VTTTALSPSQPVAFKISLKGFGAALDRIQALTK Protective antigen Brucella IalB is a protective antigen [Ref863:Commander et al., 2007]. Ifng (Interferon gamma) Mouse 15978 33468859 NM_008337 NP_032363.1 MGI:107656; UniProt:P01580 10090 ? >gi|145966741|ref|NM_008337.3| Mus musculus interferon gamma (Ifng), mRNA ATAGCTGCCATCGGCTGACCTAGAGAAGACACATCAGCTGATCCTTTGGACCCTCTGACTTGAGACAGAA GTTCTGGGCTTCTCCTCCTGCGGCCTAGCTCTGAGACAATGAACGCTACACACTGCATCTTGGCTTTGCA GCTCTTCCTCATGGCTGTTTCTGGCTGTTACTGCCACGGCACAGTCATTGAAAGCCTAGAAAGTCTGAAT AACTATTTTAACTCAAGTGGCATAGATGTGGAAGAAAAGAGTCTCTTCTTGGATATCTGGAGGAACTGGC AAAAGGATGGTGACATGAAAATCCTGCAGAGCCAGATTATCTCTTTCTACCTCAGACTCTTTGAAGTCTT GAAAGACAATCAGGCCATCAGCAACAACATAAGCGTCATTGAATCACACCTGATTACTACCTTCTTCAGC AACAGCAAGGCGAAAAAGGATGCATTCATGAGTATTGCCAAGTTTGAGGTCAACAACCCACAGGTCCAGC GCCAAGCATTCAATGAGCTCATCCGAGTGGTCCACCAGCTGTTGCCGGAATCCAGCCTCAGGAAGCGGAA AAGGAGTCGCTGCTGATTCGGGGTGGGGAAGAGATTGTCCCAATAAGAATAATTCTGCCAGCACTATTTG AATTTTTAAATCTAAACCTATTTATTAATATTTAAAACTATTTATATGGAGAATCTATTTTAGATGCATC AACCAAAGAAGTATTTATAGTAACAACTTATATGTGATAAGAGTGAATTCCTATTAATATATGTGTTATT TATAATTTCTGTCTCCTCAACTATTTCTCTTTGACCAATTAATTATTCTTTCTGACTAATTAGCCAAGAC TGTGATTGCGGGGTTGTATCTGGGGGTGGGGGACAGCCAAGCGGCTGACTGAACTCAGATTGTAGCTTGT ACCTTTACTTCACTGACCAATAAGAAACATTCAGAGCTGCAGTGACCCCGGGAGGTGCTGCTGATGGGAG GAGATGTCTACACTCCGGGCCAGCGCTTTAACAGCAGGCCAGACAGCACTCGAATGTGTCAGGTAGTAAC AGGCTGTCCCTGAAAGAAAGCAGTGTCTCAAGAGACTTGACACCTGGTGCTTCCCTATACAGCTGAAAAC TGTGACTACACCCGAATGACAAATAACTCGCTCATTTATAGTTTATCACTGTCTAATTGCATATGAATAA AGTATACCTTTGCAACC >gi|33468859|ref|NP_032363.1| interferon gamma [Mus musculus] MNATHCILALQLFLMAVSGCYCHGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQI ISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQ LLPESSLRKRKRSRC IFN-gamma plays a critical role in Th1 type immune response. It is important for protection against infections by various viruses and intracellular bacteria. Vaximmutor The experimental data demonstrated that three time vaccinations with BCG in BALB/c mice induced strong TB Ag-specific IFN-gamma immune responses in splenocytes [Ref2101:Wang et al., 2009]. IgG Mus musculus 16059 AF010213 10090 12 881411 914690 + immunoglobulin heavy chain (V7183 family) Also known as IgG; IgH; VI24H; VH7183; B9-scFv; IgVH1(VSG) >gi|94393741:881411-914690 Mus musculus strain 129/SvJ chromosome 12 unlocalized genomic contig, MGSCv37 alternate locus group 129/SvJ CATGGACTTTGGGCTCAGCTGTGTTTTCCTTGTCCTCATTTTAAGAGGTAATTTGTAGAAATAAGATCCT GCCAGTATTGTGTACAGGAGAAATAGAAAAATTTTTCTTTCCTCTATTTTGTTTTGTTTTGTTAGTGACA GTTTACAAATAAGCATTCTCTGTTGTGAGGTGCCCAGTGTGAGGTGAAGATGGTGGAGTCTGTGGGAGGC TTAGTGCAGCCTGTAGGGTCATTGAAACCCTCCTGTGCAGCCTCTGGATTCATTCTCACTGACTACTGAA TGACCTGGATCCTTCAGGCTTCAAAGAAAAGGATGGAGAGGGTGACAATAAAATTTTTCCCACTCACACC CACTTGCTGGCCCTGGCGTTCCCCTGTAATGAGGCACATAAAGTTTGCAAAACCAAGGGGCCTCTCTTCT CAATGATGGCTGACTAGGACATCTTCTGATACATATGCAGCTAGAGACATGAGCTCCCGGGATACTGGTT ACTTCATATTGTTGTTCCCCCACAACAGTTGCAGACCCTTTTATTTCTGTGGGTACTTTCACTAGCTCCT CCATTGGGGGCCCTGTGTTCCATCCAATAGCTGACTGTGAGCATCCACTTCTGTAGTTGCTAGGCCCCGG CATAGCCTCACAAGAGACAGCTATATCAGGGTCCTTTCAGCAAAATCTTGCTGGTGTATGCAATGGTGTC AGAGTTTGGAGTCTGATTATGGGATGGATCCCCAGGTATGGCAAACTCTAGATGGTCCATCCTTTCCCAA CAATAATTTTTAATGGTGGAGATAGCACCTACTATCCAGACACAATGAAGGGCCAATTCACTATCTCTAG AGATGATGCCAAAAACACACTTTACCTGAAAATAAACAGTCTGAGGTCTGAGTACACAGCCATGTATACA TATATTCCTAAGCTTGAGTAAGTGGACTTAAACTGATTCCATCACAACTTGCATGAGATGGATATTCCCC AGTGTTAAGACCTGTCACCATCACTGTCAATCAGAAGACAAAGTTTATGCACAACAAAACAAAAAAACCA AAAGCAGAGGCCTCCAATTACAAGTAATAGACCCAGACCCACAGTCTCTGAAAACTGACTGTACAGTTGG ATCCAGTCTTTTACTTCTTTTCCCTGGATTTTATATTACTGAGGAAATGAGGAAAGCTCTACAATATCTG TTCTCCATAGTGCTCAACACCTCCAAGCACAGGTTACCCATATTCATGCCTGCCTTCTGCTACACTTCTT GTCTTGTAGACTACTTCAACCTATTTTGTACTCCAGTTAATGAAACTCAAGTCTAGCAGCCTGTCACTGT TTATTCTAAAGTATTATGAACAGGTGACCTCCCATCCTTCCCCAACGCAATAATCATATTTAGGAATTTG AGGTTTTATGAGATATGATCTCAGGGTAGAGAGAGAAAGCAAACTACATAGAAATATAGACTGACATAAA TCAAGACTTGCATAAGCTAGTCCCCAAGTTCCATGTCCCTAAGTGGCAAGGACTATCTTCTGAGCCTAAT GAGATGAGATCCAAATCAAACCTCCTGGGTCTTTTTAGATAAACATGTGAGATCAATAGACTAAATGCTT TGGCTGGGCTTCCTTGCAATCCAATTCCCAAACAAAAATGGATCTGGCTCCACAGACACCACAAGAATAG TCTTAAATAGTTCTTTAAGTAGAATGTCTGATCACTACGAGCCCAATTCCATCCTAAATACTCTTCTGGA TTATACATAAATAAAAATTGAACATAGGGCATGGGGCACTGATCTCCCTGTGCTACATGAATGGGGGCTC ATTTACTAAATGTTCCCATTTTTCTTTCTAGCGCTGCACAGTGCAAATCCTACAACTTCCTGTTTATCTA CAATGTGAACTCCAAACAGTACAAGAAAAAACGTTCCTTATGTTCCTCTCCAGGTTCTCCAACAAGCACA GAGCAACCTTCTGTCACCAGGACGGAAAACTGGAAACCTTGCTCACTGCTTCCTTTTATTCCCTCGGGAA CCTCCCCCAATGCAAAGCAGCCCTCAGGCAGAGGATAAAAGCTCACACAAAGACGAGAAGCCCCATCCTC TTCTCATAGAGCATCCATCAGAGCATGGCTGTCCTGGGGCTGCTTCTCTGCCTGGTGACTTTCCCAAGCT GTAAGTGTTTCAGGGTTTCAGAAGAGGGACTAAGACATGTCAGCTAGAAGATGTGTGACTAATGGTGATG TTGCTTGTCCCCAGGTGTCCTGTCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCA CAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACTAGCTATGGTGTACACTGGGTTCGCC AGCCTCCAGGAAAGGGTCTGGAGTGGCTGGTAGTGATATGGAGTGATGGAAGCACAACCTATAATTCAGC TCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTC CAAACTGATGACACAGCCATGTACTACTGTGCCAGAAACACAGTGTGGGAAGTCCAATGTGAGCCTGCAC AAATACTTCTCTGCAGGGATGATCACAACCAGCAGGGGGCGCTGATGACCCAAAGGGACTTCCCAGGATC TCTTCTGGAATCTAGGGAGTTCTGGCCTGTGTCTATCAGCATGTGTTTCAATGTTAGAGTTGTGAGTTTT CCTTCCAGCAACAGAGATATTTTAGAGCCCACTTTTCATGGTCATTCTACTAAATTTGTTCACATAGTGG AAAGATTTGTTAAATGATTCTATAGTCTAATAGGGTCAAACAAAAACAAATAATTGAGTTCATATCTACC AAAAAAAAAAAAATTCCCTCAAAGTGGACAACTGTGTAGGTGAGGAAAACCAGGGGGATTTGGGAATAGA TTATTTCTCTCCCGACTGTGGTGTTAGTCACCTTTCAGCCATTTACTGTATTTAATTCATGTTTAAAGTT AATTGTATTCATCTTCCCTAACATGAAGTTTTCAAACACATGTCCACAATTTAATGATGACACAGTTATC ATTGTGAATGATAACTCATAAGATCTCTCTCCATATATGAAACACACGATATTCTGTTATTAGCTATAGC CAACAAAGTTACATATCATTATTATTACTGAAATAATTCTTCCCATCTAACTGAATAGTTTTCTCACTCG ACACTGCCTATGCTGCGATCAGCCTGCTCTAATTTTACCTTTGCTCAGAGCACTGCTTTCTGTTTCATTT GATAAGATGGTGCCTGGTTGTCACTGAAAATGTGTCCTCCAGTTTCACAGTGATTAATGTGATTTTCAGT ACATTGAGAACAGAGCCACAGCAAAAATGAGATGGAATGTCATCATTTTTACGCCAGGTGCTGCAGAAAT AGCTAGGTGGTTAAGAGAGAATATTGAACTTTGAGGGGTTCAGATTAAATTCCTGATACCCACAATGTGC ACCTCATAACCACATGTACATATAGAAAACTCAGTATAAATGTGGCCTCCGTGAGCACCACACTACTCCC ACAAACACATATACACATAATTAAAAGTAAAGATTTTAGAAAAATGGCTAATCTAATGATAGGAAGTAGT CAAAAAGAGAGTTCTTTTGTCATGTACATATGTGTAGCAGACTTAAATGTTAAACATTCAAGAATACATT CCTCGATCCACATTAAAATTTTGCAAAGAGTAGCACAGACGGTGGCAATTGCTAAACTTATATCTAGAAA CACAATTATGTGTGGTGATATTTAATTACACATTTATACCAGGACATATGACAATATGGAAACCAAACAT GTTGTATCCACATGCTCTAAGGAAAACTAAATGGAGTGTGATAAAACCAAAGAAAATGTGAATATGAAAT ATTTTTCCAACTCTGCATCTTAAAACGGTTTCTTTCATGTGTCATATCTGCTATGAGGACTTTCTTCTGC CCATGTCCAACTCCAGAGCATGCCACAGCAGGAAGACCTACAGGTATTACTTCTCTGCACCCAGGAAAAC CACCTCTGTCCTGACCCTGCAGCTCTCAGAAGAGCCCAGACCTTCATTCTCAGGCCCTCATCCAGTAATC AGCACTGAATACAGAGCACTCACCATGGACTTTGGGCTCAGCTTGGTTTTCCTTGTCCTTATTTTAAAAG GTAATTCATAGAGATAAGATTCTATCTGTTTTGTGTACATGAGAAACAGAAAAATTGTATTGTTTCTCTA TTTTGTTTTGTTTTGTTAGTGACAGTTTCTGACTCAAGATTCTCTGTTTGAAGGTACCCAGTGTGAGGTG AAGCTGGTGAAGTCTAAGGGGAGGCATAGTGCAGCCTAGAAGGTCCATGATACTCTACTGTGCAGCCTCG GATTCACTGTAAGTGACGACTGGTTTGTCCGTGTTTGCCAGGCTCCAAAGAAGGGGCTGCAGTGGGGGAT GGGAATAATTTTTCATGGTTGTGGTAGCCCCTCTTATGCAGACACCTTGAAGAAGTGGGTTGGACGTAAC ATATTCAGAATCAATATTTAAAGATTCTAATCCTTGAAGATATCACTTTTGACCAAGTATATATGAACCA TGTTACTGAGGTTTATGGAGGTTTGAGTATGTTAGGTCCATGGATAGGGAAAATATTAGGGGATTTTAGG AGTAACTGAGGCTTGTTGTAGGAAGGACATCACTGTAGGGGTAGGCTCCGTGTTCCTATCTTCAAGCTCT ACCCAGTGCAGAATAGAGCCCTCTTCTGCCGGCAAGTGGAAAGAGTTTCTCTTCCTGGCTGCCTTCAATC CAAGTTGTAGAATATCAAATCTCCTAACACTATGACTGCGAGCATGCTGACATGCGTCCCACCATAAAAA TAGACTGAACCTCTGAAGCTTTAAGCCAGCCTCTAGTAACTGTATTCTTTAATAAGACTGAACTTGGCTA TGGTGTCTTTTCACAGTAAAATGGAAACATAGACAAGGTTCTAACTCTCTGTACTAGAGGACCATCTCTA TGTCCTTGGTGTTGTATGTAAATTATTGAACAAATACTATAGACAAGTTGTATGGCCAAAAGTCCATCCC CTTCAACAATCTACTATCTGGAACCATATAGATTAGATTCCTTTCCTGCACTCATTTCCCTTACTTGCTG AGACATTTTGAAGACATGCTCAGAATCCCTGAACTTCCTGCTGAAAAAAAAATACCCCTCCAATTTGAAG ACAGTTCTCTTCCAAAATTCATCATAAATACTGATCCACATGTCCAGGCATATCATGTAGTTTTAAAAAA CAAACCAATCAATGCTAAAGTGGGTAAGTGCCTACATCATTGGTTTATGTCTGTTCATAACCTGGTATAT AGCTAGAGACCAGGACTGCATGTTTCTCCTAGGCCACACCTCTCCCACAAATGCTGGCTCTGCCTCTCCA GAATTCCAAGATCATGGATCTCTGGGTCCCCAAGGAACAATAAGTGTGCACTCTTGCCAGCCTGTACACT ACTGCACTAGCTTCTCCCCCTGGAGCTTGGTCATTGCTCAATCTTCCCTTTCCAAAGCCTGGCCAAATTC CTCCTACCTTTTGTTCTTTCAGTCAGGCCACCTAAGTCATCTTGAATGAAAAACAACACAAAATCTGAAC TTAAAATCAACAAATAATACAAGTGCTGCGCACAAAATCAATCATTCTAAACTCATCAACTATTTTATGA TGGAAATCTTCCAACATACAAGCTACCACCAGGTCTAAGAATTACTCATCTACATGTTGCTTCCTCTCAC TCACAGCCTAACCATAAAAGGGCTGTTTCTTCCTCCCATGTCCCCTCTTTTGCCTTCAGAAGCAGAAGCT CCACCTCCTCCTCTTTGCCCAGCAATTGGCTTCTTGTCGTCTTTATTATCATATTAATTACTTAGGGGAA AATCCCGTGTAGTGGCTATTCCTGGTTGTCAACTTGACAATATTTGGAATGAACTACAATCCGGAATTGG AAGGCTCACCAGTGACCCTTATCTGGAGGCTTGGAGATCCTTATCTGGATCTTGGTTTGAAGATCTTGAG CCATAGGGGCTATGGATTCCAGAAGATTGAATCTCCGAGTTTAAGGAACACACCTTTAATCTGGGCTACG CCTTTCATCTGGGATTAAAGGTGTGGCGGAACACACCTTTAATCTGGACTACACCTTCTGCTGGAGACAA TATAAGGACATTGAAAGAAGGGAGTCTAGCTCTTGCTCTTGCTCCTTCGCCTGCTTGCTGCGTGAGACTG AGTAACTGCTAGATCCTTGGACTTCCATTCACAGCTGCGACTGAACAATTGTTGGGAATTGGGCTGCCGA CTCTAAGTCATCAATAAATTCCTTTACTATCTAGAGACTATCCATAGTTCTGTGACTCTAGAGAACCCTG ACTAATACAGAAGTTGGTACCAGGAGTGGTTCTAGAGTAACAGAAGTACAAGGATGAATCTTTTAAAATA CTGGAATTGGCTTGTTGATCCACCAGCACTTTCAACTATTGAAACCTCTCCAGATTCTCTCCCTCCTGGG AGCTCAGAGAATTTTGAAGACCCATGGTTGAAACTATATTCCGAACTTAAAGAAGCAAATGCCCTTGATT TTCTTAATGAATTAGGTGATTCAGTGCACAAAGCTTTCTACAAGATGGGGAAAAAATTGGAAAATGATTT TACTGGCTGGCTGCTCTTAGTATCTGTGGAAAAAATGATGAATGAAAGGAAGGAGTTGTGTGATAAAATC GAAAGGCTCCAGACACAAGTAAACGATCTAAAAGTTGCTAAGTGTGTCCTTGAGGAGAATCTTCTCTCTT GTAGCAATAGAGCTCAAGTTGCAGAAAATCAAACAGAAACTCTCATTGTAAGGTTGGCTGAACTACAGCG AAAATTCAAGTCTCAGCCTCAGAGTGTGTCAACAGTTAAAGTAAGGGCTCTAATTGGCAAAGAATGGGAT CCTACAACATGGGACGGGGATGTGTGGGAAGACCATGTTGAAGCTGAGAATTTTGAATCTTCAGATTCTC AAGGGTTTGCCCCACCTGAGGAAGTAGTACCCTCAGCCCCACCCCTTGAAATAATGCCTTCCCCACATGA GGAAATTAATTTTGCAGAGTCTGATAAACCAGCAATGATTTTCACTACTGATGTTTCTCAAGGCCCACCA ATAGTTTCTTCTAGACCTGTAACCAGACTCAAAGCAAAACAGGCTCCTAGAGGGGAGGTAGAAAGTGTAG TCCATGAGGAAATTCGCTACACTACTAAGGAGCTTAATGAGTTTGCTAATTCATTCAAGCAGAAACCTGG TGAATATGTGTGGGAATGGATTTTAAGGGTGTGGGATAAGGGTGGAAGGAACATAAGACTAGAGCAGGCT GAGTTTATTGACATGGGTCCTCTGAGTAGAGATTCTAGGTTTAATACGGAAGCTCGCATAATTAAAAAAG GTGTCAAAAGTTTGTTTGAATGGTTGGCTGAGGTGTTTATCAAAAGATGGCCTACTGGAAATGACTTGGA GATGCCTGATATTCCGTGGCTTAGTGTTGATGAAGGGATTTTAAGACTTAGGGAAATTGCAATGCTAGAG TGGATATATTGTGTAAAGCATAATTGTCCACAATGGGAAGGTCCAGAAGATATGCCTTTCACTAGCTCTA TAAGACGCAAATTGGTGAGAGGGGCACCAGCACATTTGAAGGGTTTTGTTCTTTCCCTTTTCCTTGTACC AGATCTTAGCATTGGAGATGCTTCTGCTCAATTAGATGAATTAAATTCACTGGGTTTAGTTGGATTCCGA GGTAACAAGGGCCAGGTGGCAGCATTGAATCGCCGGAGACAAGGTGATTCTAGTTATTATAATGGACAGC GTAGACAAAAGAATGTTTATAATAACATACCCAGCAATGGTCAGCACAGGAGAGGTGAAATTTATAATGG CATGACTCGGTTGGACCTTTGGTACTGGCTAACCAATCATGGTGTTTCCAGGAATGAAATACATAGGAAG CCTACTGCATATTTGTTTGATCTGTATAAGCAGAAAAATTCTCAAACAAATGAAAGAAAGGCTACATTAG ATCATGGTAAACAGCAATCTCGGCCAGTGAATCAATTTCCAGACTTGAGACAGTTTGCAGATCCAGAACC CCTTGAATGAAGGGGTGGCCAGGTTCCGCTGAGGAAGGATCTTGATAAGACACCCAAAGGTTTTGCTGTT ACCCTTTCTCCAGTTCTTCCCCAGAGGGACCTAAGGCCTTTTACAAGGGTAACTGTACACTGGGGAAAAG GAAATAATCAGACTTTTCAGGGTCTGCTGGATACTGGTTCTGAGTTGACACTGATTCCAGGGGATCCCAA GAAACATTGTGGCCCTCCAGTTAAAGTAGAGGCTTATGGAGGGCAGGTGATTAATGGAGTTTTGACTGAT GTCCGACTCACAATAGGTCCAGTAGGTCCCCAGACACATCCTGTGGTGATTTCCCCAGTTCCAGAATGTA TAATTGGGATAGATATACTCAGAAATTGGCAGAATTCTCATATTGGTTCCCTGAACTGTAGAGTGAGGGC TATTATGGTTGGAAAGGCCAAATGGAAACCTTTAGAGTTGCCTCTGCCAAAGAAAATAGTGAATCAAAAA CAGTATCGTATTCCTGGAGGCATTGCAGAAATTACTGCCACTATCAAGGACTTGAAAGATGCAGGGGTGG TGGTTCCCACCACATCTCCGTTTAACTCTCCTATCTGGCCAGTGCAGAAAACAGATGGATCATGGAGAAT GACAGTTGATTATCGAAAACTAAATCAGGTAGTAACTCCAATTGCAGCTGCTGTACCAGATGTAGTTTCA TTACTTGAGCAAATTAACACATCTCCTGGCACCTGGTATGCGGCTATTGATCTGGCAAATGCCTTCTTCT CAGTACCTGTCCATAAGGACCACCAGAAGCAATTTGCTTTCAGTTGGCAAGGCCAACAGTATACTTTCAC AGTTTTGCCTCAAGGATATATTAACTCTCCTGCCCTGTGTCATAATTTAGTTAGAAGGGATCTTGATCGT TTGGATCTTCCACAAAATATCACATTGGTGCACTATATTGATGACATTATGCTGATTGGACCAAGTGAGC AGGAAGTAGCAACCACTTTGGACTCATTGGTAACACATATGCGTATCAGAGGATGGGAAATAAATCCAAC CAAAATTCAAGGACCATCTACCTCAGTGAAATTCTTAGGAGTCCAGTGGTGTGGAGCATGCAGAGATATT CCTTCTAAGGTGAAAGATAAGTTATTGCACCTGGCCCCTCCTACAACCAAGAAAGAAGCACAACGTTTAG TGGGTCTATTTGGATTCTGGAGACAACACATCCCTCACTTGGGTGTGTTACTTAGGCCTATTTACCAAGT GACTCGGAAAGCTGCTAGCTTTGTGTGGGGCCTGGAACAGGAGAAGGCCCTTCAACAGGTCCAGGCTGCT GTGCAGGCTGCACTACCACTTGGACCATATGACCCAGCAGACCCGATGGTACTTGAGGTGTCTGTGGCTG ATAGAGATGCTGTTTGGAGCCTCTGGCAGGCCCCTGTAGGTGAATCACAGAAAAGACCTTTGGGATTTTG GAGCAAAGCTCTACCATCATCTGCAGACAACTATTCTCCCTTTGAAAAACAGCTCTTGGCCTGCTATTGG GCCTTAGTGGAAACTGAACGTTTGACAATAGGACACCAAGTTACTATGCGACCTGAACTACCCATCATGA GCTGGGTACTATCAGACCCTGCAAGTCATAAAGTGGGACGCGCACAGCAGCAGTCTGTTATCAAATGGAA GTGGTATATACGTGATCGGGCCAGAGCAGGTCCTGAAGGCACAAGCAAGTTACATGAAGAAGTTGCTCAA ATGCCTATGGTTTCTACTCCTGTTACACTGCCATCTGCTGCCAAACATGTGCCTATAGCCTCATGGGGTG TTCCCTATGATCGACTGACCGAAGAGGAAAAGACTAGAGCCTGGTTTACTGATGGCTCTGCACGTTATGC AGGCACCACCCAGAAGTGGACAGACAGCTGCAGCATTACAACCCCTTTCTGGGACAACCCTGAAAGACAC AGGTGAAGGGAAATCTTCACAGTGGGCAGAACTTCGGGCAGTACACATGGTATTACAGTTTGTTTGCAAG AAGAAATGGCCAGATGTACGATTATTCACTGACTCATGGGCTGTAGCCAATGGATTGGCTGGATGGTCAG GGACTTGGAAAGATCACAATTGGAAAATTGGTGAGAAAGACATCTGGGGAAGAAGTATGTGGATAGATCT CTCCAAATGGGCAAAGGATGTGAAGATATTTGTGTCCCATGTAAATGCTCACCAAAAGGTGACTTCAGCT GAGGAGGAGTTCAATAATCAAGTGGATAAGATGACCCGTTCTGTGGACAGTCAGCCTCTCTCCCCAGCCA TCCCTGTCATTGCTCAATGGGCACATGAACAAAGTGGCCATGGTGGTCGAGATGGAGGTTATGCTTGGGC TCAGCAACACGGGCTTCCACTCACCAAGGCTGACCTGGCTACAGCTGCTGCTGATTGCCAGATCTGCCAA CAGCAGAAACCAACACTGAGTCCCAGATATGGCACCATTCCTCGAGGTGACCAGCCAGCAACCTGGTGGC AGGTTGACTACATTGGACCACTTCCTTCGTGGAAAGGACAGCGTTTTGTTCTTACTGGAGTAGATACTTA TTCTGGTTATGGATTTGCCTTTCCTGTACGTAATGCCTCTGCTAAAACCACCATTAACGGACTGACAGAA TGCCTTATCTATCGTCATGGTATTCCACACAGTATTGCTTCTGACCAAGGAACTCATTTCACAGCCAGAG AAGTACGACAGTGGGCCCACGATCATGGAATTCACTGGTCTTACCACATTCCCCATCATCCTGAAGCAGC TGGTCTGATAGAAAGATGGAATGGCCTTCTGAAGACGCAGTTACAGCGCCAATTAGGTGGTAACAGCTTG GAAGGCTGGGGTAGAGTTCTTCAGAAGGCAGTATATGCTTTGAATCAGCGCTCGATATATAGTACAGTTT CACCCATAGCCAGGATTCATGGGTCCAGGAATCAAGGGGTGGAAAAAGGAATAGTTCCACTTACTATCAC TCCTAGTGACCCTCTAGGAAAATTTTTGCTTCCTGTCCCCATAACTCTAGGTTCTGCTGGCTTAGAAGTT TTGGCTCCAGAGAGGGGAGTGCTCCTACCAGGAGCTACAACAAACATTCCATTGAACTGGAAGCTCAGAC TTCCCCCTGGTCATTTTGGGCTTCTAATGCCCTTAAACCAACAGGCTAAAAAAGGAGTAACAGTGTTAGG AGGGGTGATAGATCCAGATTACCATGGGGAAATTGGATTACCTCTTCACAATGGTGGTAAGCAAGATTAT GTCTGGAGTGTAGGAGATCCCTTAGGGCGTCTCTTAGTACTACCATGTCCTGTGATTAAAGTCAATGGGA AACTACAACAGCCTAATCCAAGCAGGATGACAAAGGACACAGACCCATCAGGAATGAAGGTATGGGTCAA TCCTCCAGGAAAAGAGCCAAGACCTGCTGAGGTGCTGACTGAAGGAGAAGGAAATATAGAATGGGTAGTA GAGGAAGGTAGTTATAAATACCAATTAAGGCCACGTAACCAGTTGCAGAAACGAGGATTATAAAGTAATA TGAATGCCCATTGTAAATTTACTAATGCGTTTGCGATTGTACGAGGGATAGTTATATCATGTTAGGCGTA TTTACAAACTTGTTATTGTTTTATGTGAACATGAGATATTATTTGTGTCAAGTTGACAAGGGGTGGATTG TAGTGGCTATTCCTGGTTGTCAACTTGACAATATTTGGAATGAACTACAATCCGGAATTGGAAGGCTCAC CAGTGACCCTTATCTGGAGGCTTGGAGATCCTTATCTGGATCTTGGTTTGAAGATCTTGAGCCATAGTGG CTATGGATTCCAGAAGATTGAATCTCCGAGTTTAAGGAACACACCTTTAATCTGGGCTACGCCTTTCATC TGGGATTAAAGGTGTGGCGGAACACACCTTTAATCTGGACTACACCTTCTGCTGGAGACAATATAAGGAC ATTGAAAGAAGGGAGTCTAGCTCTTGCTCTTGCTCCTTCGCCTGCTTGCTGCGTGAGACTGAGTAACTAC TAGATCCTTGGACTTCCATTCACAGCTGCGACTGAACAATTGTTGGGAATTGGGCTGCCGACTCTAAGTC ATCAATAAATTCCTTTACTATCTAGAGACTATCCATAGTTCTGTGACTCTAGAGAACCCTGACTAATACA TCCCTACATACAGGAAACTTGATGTATAAGTAAAGAAAATAGTAAATCTAAAAATTTCCTTATCAAATAC ATGCAAGAAATCCAAAATACCATGAAAAGACCTGATCTAAAAATAGTAGGAACTGAAGGTGAAGTGTCCC AGCTCTGAAAACTAGAAAATGTTTTCAATATTTTTCCTAACAGTGAAGACATGGATCTAATGAGGCCACC TCTTATAGCCATGCAAGACTGACAGTGGAGGGATAAGGACACCAATCCACCCACAAAACTTTTGACCCTA AATCAGTTTTATCTAAAAGCAATGCAGGGGCACAAATGGAGCAGAGACTGGAGGAATGGTCAAACAATAA CCTGTCATATCTGAGACCCACCTAATTGGCATACACCAGTCACTGACATTATTAATAATGTTCCATTATG CTTACAGGCATTTCTCTAGCATTACTATTCTTGGAGAGACTTCACACAGCATCTTATTGAAACAGATGCA GACACCCAGAACCAAACATTGGATGGAGATTGGGATCCTTAAAGAAGAGTTGGGAGGATAATTGAGAGAC CTCAAAGGAATAGCAACCCCATAGAAAGACACGAACAGGGTCAGTAAACCTGGACCCATGGGGTATCTCA GAGACTGATCACCAACCAAAAATCACAGAGGGCCTGGATTGATTCCCCTGGCACACATGTAGCAGAGGTC TGCCTTGTCCTTCAGTAGGTGAAGATGAGCCTAAGGCTTCAGAGACTTGATATATCAGCATACAGCAACA CCCAAGTGTGTCCCACTATCTCAGAGGTGAAGGGGTGGAGATGGGCAGAAGGGTCCTGATAGGGGACAAC TGCAAAGAGGCAATATTTGGGACAGAAAGAAAGGAAGAGAGAGGAAGATAAAGAGAGAGATGAAGGAAGG AAACAAACAAACCAAGATGAAGAGAGAGAAAAGGGGGAAAATAGCCTTGAATGGTTCAGGGATTCAAAGG GACATACCTAAGCATGGTAAAACAATGCACATAATGGCAGCAAGTAGCTAACACTGAATTAAATAGAAAG ACACATAAAGCAGTTCTACTAAAGTAAGAGACAGAGTTGCCCAATCTCACCCTATTTATTCAATAATAAT GTACTTGAAGTTCTAGCTAGGGCTGTAAGACAACTCAAGGAGATCGGAGGATACAAATTAGAAAGAAAAA TTCAAAGAATTGTAATTGGTAGATGATAGGATAATATACATAAATGACCTCAAAATTCTTCCAGAGAACT CTGAAAGCTGATAAATACCTTCAGCAAAGTGGCTAGAAACAAAATTACTTCATAGAATTCAGGAGCTATC CTTTATAGAAAAGTTAAAGTGGCTGAGAAAGAAATTAGGAAAATCACATTCTTTGCAATAACCAAAAATA ATATGAATTAGCTTGGTGTGACTCTAATCTTGCAAGTGAAAGATGTGTGTGACAAGAGCTTCAGGACCCT GAAGAAAGAAATCAAAGAACTCAAAGATGCAAAGTACTCTAATGCTCATGGATAGGCAGAATTAACATAA TGAAAATGCCAAATTTAACAATTCAATCTACAGATTCAGTGGAATTCCCATTAAATATCCAACCCAATTT ATTACATTCTTGAAAAAGCAAATCTCAACTTCATATAGAAAAACAAGAAATTGAGGGTAGCTAACAAAAT CCTGAACAATGAAAACACTTCAGGAGAATTCACCATCCCCTACCTCAAGCTGTATTTTAGAGCAATAGTT ATTAAAACTGCATGGTATTTGTATAGAAACAGATATGATGATCAATGTAATTGAATTGAATACACTGACA TAAAACCACACTGTTATGGACACTTGATTTTTGACAAAGAACCCAATAATCATAATAAAAATCATAATAA GAATGCATCCCCCAACAAATGGTCCTGATCTAAATGCAAATAGATTCATATCTATCATCCTGCACGAAAT TCAAATCAAAGTGAATTGCAGACTTCGACCTAATACTGGATTAACTAAATCTAAATGAACAAAAAGTAGA GAATAGTTTTGAACTCATTGATGCAGGATTCAATTTCCTGAACAGAACCACAATGGCTCAGGCTCTAGGA TCATAAATTGGTAGATAGGATCTCATGAAACTGAAAAACTTCTGTAAGGCAAAGGCAATAAAACAAAATG GCAACCTACAGATTGGAAAAAAAATAATCTTCAGTATCCCTAAATCCCATTGAAGGCCAATATGCAAAGT ATATAAAGAACTCAAGATGTTATCCTCCACAAAACCAAATAAATCAATTAAAAATGAACTACAGAGCTAA CAAGAGAATTCTCAACAGAGGAATCACCAATGACTGAGAATCACTTAAAGAAACATTCAATATTTGTAGT CACCAGAGAATTGCAAATCAAAACTACCCTGAGATTCTACTTTATACCAATCATAATGGTCAAGATAAAA ATTCAAATGACAGCGCATGTTGGTGAGGATGTGTATACTTTTGCATTGCTGGTGAGGAAAACAATCTGGC AGCTCCTCAGAAAATTGTAAATAATTCTACCTGAAGATCCAGCCATACCACTCCTGTGCATATACATAAA ATGTGCTCCACCATACCACTAGGAGATATGTGCCACTATGCCCATAGCAGCCTTATTTGTTAATAGCCAA AAGATGGATACAACCCTGATGTGCCTCAAACAAAGATTGTATATAGAAAATATGGGTTCCCTTACACAAT GGTATTCTACTCAGCAATTAAAACGTGAGAACATCATAAAGTTTTCAGGCAAACTGATCAAATGAAAAAG TATAATCTTGAGTGAGGTAACAGAACCAAAATGACCTGCATGGGATGTACTCACTGGTAAGTGAATATTA GACTAAAAGTATAGAATATCAATGATGAAAGCCACAGACCATAAAGAGTTTAATAAGATGGAAAGCTCAA GTACGGATTCTTCAATCCAATATAGAAAGAGGGACAAAATAATCAGGGGAGGCAGAGGGAGAGAGAGACA TTTGGTGGAAATGGGAGAAGAAGGGAGAAAGGCAGAAAGGATCAGGCGTTGGGGAACACTGGAAAGCAGC CCAGGGGTCCTGGTAAATGGATTAAATATTCAGCTGAATGGAGGTGGGAGGCAGGGGGAAGCTCTGCAAA GTCCAAGAGACCTGGGATGTGAGAGGCTCCCCAGATGATAATCTTAGCCTTCATGCCTAACAGTTGTAGA TAAACCCTGAAGAGATCACTTCCAATAGATGCAGAGGGCCCTAAGTGGTTGGATGGAGTCCCCCAACTTA CCTCAAAATTGTCAATACTCATAGAAATTAGGACAATGAAAATCAAAACACCCTGAGATTGTATCTTATG TCTGTCACAATGGTTAAGACCAAAACCTCAAGTGATGGCTTGTGCTGGCAAAGATTCAGAATAGTAAAAG TCTCCCCATGGCTTGTGGGAATGCAAACTCTTACAACCTCTTTGGAAGTATATTTGATTGTTACCTGGTA TAAAATGGCAAAAAAAAAAAATGGTTGAAGGGGGACTAAAAAAGGAAGAAAGGGGAGAACTATGGGATGC GAAACAAGAAAGTTTGTTGCAAAAGAAATATGTTTCCACTGCAAACCCTGAGTCTCAGACAGAAGGGGAC CTGGAATTCTTCAGATACAAAGAATCTCTAAACCCTGAGGACATTCTATCACAAATAAGTAAAATTCAGA AAATTCTGAGTGCTCCCATCACGGAGATGAATCTGCTATGAACAGCTCATAGGTGTGACCCTCTACAAAA GCCATATTATTGAAAAGCCACATTGTGCCCAGACTTTGGAAAGACTGAGCTCATATCCTGAAATACAGTT ATGTGTGGTTCTATTTAATTACACATTTACACTAAGAAAACATGGCAGTATGGGAATGAAGCTTGTTCTG TACACATTAACAGAGGGAAACTAAACAAAGTATGGTGAATCCCTAACCAAAAGTAAAAAAAAAAAAAGAA AGAAAAGAAAATAAAAGTGAAACTACAATATGTTTCAAATGCTGTAACTGAAATCTGGTTTTTTGATGCT TATATCTGGTATCATCAGTGACTTCAGATTTAGTCCAACTCCAGAGCATGGTATAGCAGAAAGACATGCA AATAAGCCTTCTCTCTGCCCATGAAAAACACCTCGGCCCTGACCCTGCAGCTCTGACAGAGGAGGCCAGT CCTGGATTCGATTCCCAGTTCCTCACATTCAGTGATCAGCACTGAACACGGACCCCTCACCATGAACTTG GGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTAATTTATTGAGAAGAGATGACATCTGTTGTA TGCTCATGAGACAGAAAAATTGTTTGTTTTGTTAGTGACAGTTTTCCAACCAGTATTCTCTGTTTGCAGG TGTCCAGTGTGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTC TCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGA GGCTGGAGTGGGTCGCATACATTAGTAATGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCG ATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCCGTCTGAAGTCTGAGGAC ACAGCCATGTATTACTGTGCAAGACACACAATGAGGAAATGTTACTGTGAGCTCAAACTAAAACCTCCTG CAGAGCACCCAGGACCAGCAGGGGGCGCAGAGAGCACATGGAGTTCTGATTCACAGAAGAGTTACAGCCT GTACAATTAGACCCAATCTTCAACAAACCGTCAAAATATTCGATCCAAAATTGTTCCTGTCTAAAAGTAA TTCAAGGACAAAATGGACCAGAGACTGAAGAAATGGCTGACCTGTGACCCTCCCAACTTTGGATCTATCT CATAGGCAGGTACCAAACCTTGACATTTGTCACTGACACTGTATTGTGCTTGCAGACAGGAGCATAGCAT GGCTGACCTCTAAGAGGCTCTGCAAGCACCTGAATGAGACAGATGTAAATAGAGTGTACAACTGGATTAA GTTTGAAGACTGCAATGGAAGAGTTAGAGGAAGGAATGAAAAATCTGAAGGGGATCACAACCCCAGAAGA AGACCAACAGTGCCAATTAAACTGGATGCCTGGGAACTCCCAGAGACTAAGCCACAAACCAAGAATCATG CAGGCTGGTCTGAGAATCCTGGCACCCAAGTCTCAAGGAACTGCCTTTTCTTGGCTCAGATGGGAGAAGA TGAGCCTAAAGCTTTAGACTTGACGCCCCAGGGATGGGCAATACAAAGTTGAGTTTCTTGGGAAAGTGAA GAGGATGGTGGGAGGAGGAATAAATCTGGGAAGTGGGACTGGTTGGGGACAACATATTGGTCCTAAATTT ATGAAATAATTATCTAATTGATAAAAAGAGTCCTTGGGGTAATGGAGGGCTAGACTCCTCTGTGCCTAGT TTGTAACTCTACAGGGATCCTCTTTAAAAGAATAGGGTGCACATAGAGTATATGTGTGTGACACTAATAC AGGGGTAAGTGTTTCTGTAAGAACTTTATGAATAAACTTTATTAAAACATCAAAAAGTATAGGTTGTAGC AACCCTTGACCTGTACGAATGTTTATAAAACTTTCTATTTTCCTTAATTATATCTGTTTTGCATTTGTTT ATTTATTTATTTACTTACTTACTTATTTATGATTTCACTGTGTCTTTCTGTAGTCCTGTATGTGTTTATT TGTCTCTTTATTGCTCTGTTTCTGTTTCTTTCTCTCTGTCTAGTATTGTATCTCTCTGTGTCTCTATGTC GCCGATTGTGTCTGTGTGTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCATATTGAAA TGAGATCTCTTCTTCTATCAGTGTACATAATGACTTTGAAATAAAGTCCATCACTGGGCTTGGATATCAC TATCTTGTTAGAATGGTTGCCCAAAGTGTTCCAACCATCACACTGTTTCTACATATAATAGATGGGTTTA TTGATTTACTACCAAAACTACCCCATCTTAAGTTATGGATATTAAACACAGATCCTCATGGTGTGTTAAG AACTTGAAGCACAGAGCTATATGTACAGCCCACTAAAAATGACTTAAATATAAGATAAACTCTGGCTTGA ACAAAACACAGAGCATCACACGAGGCAGAAGTGAATCTCTATGAGGACCAGGATCACATCCCAAAGAACC AAAGAACACTTGAGACTGTGGAACCCTAGAGTCAGGGCTGACACTCAGTAAGATTTACTTCATAGAGCAT GATGTGGGATTCAAGAAGAAATCAGGCAGGGCCTGTGACCCATGAACCAGATGACTTCCAGTCTCTCCAT CATCCTCACACAAACCATAGGCAACAGCTCAGCACTGACTCACCTGTGATGCTGGTGACAAAATGTGTGC AAAGCCTCAGAACTGAGACGTGAGGCCAATGTTGCTACCGACACTCAGTGAGCTTCAATTTGCTGCAAAC TGCTCCAAAACAACTCAAAGGGTAACTTCTTCTTTTTCTATTTGTACTTGTGATGCCTTGGTAGATATAG TTCCCCAATAATTTAATAGGACAAATCATTTTTCCTTTTAACACATTAATCCCCTTTCCTTCCCCCAAGA TTCTAAAATTTTTCTTTAACACTGATGTCTGAGAGATATGGCATAATAGATGTTTGTCTTATTCTGAGTT GTTCCTTATCAAAAGAAATCAGCTGTGGCACTTTGAACCTAAATATAACAATATTTCACAAAGGCATTCT GGGAATATATAGATGGTATTGCTACAAAAGAATAGGAGGATCTATGGCCATACCACCCTTAATGAACCAG ATACTATCTAAAAGATTAGGACAGCTGGGCATGTTGGCACAGGCCTTTAATCCCAGCACTAAGGAGGCAG AGGCAGGCAGATTTCTGATTCGAGGCCAGACTGGACTACAAAGTGAGTTCCAAGACAACCAGGGCTATAC AGAGAAACCCTGTCTCGAAAAAACAAAACAAAACAAAACAAGAGTAGGAGGGTATTAGATGCGAGGTGAT GATGGCCATTCAATCAAACCAGAATGTGACCTGAGAAAAGGTCTAAGTTTGTCAGCTTGTCCTGGAGATG TGTTGCCTCTGATAAATGCAGAGCTGGAACTGCAATACATTGGATTCCTGTGAGTTACGGAGAAATAAAA GGAATAGCCTCTGTTTCCTTTGGTTCATCAAGTTGGCAGTGAGCTAGAAGATGATACTGGGAACCATCAC ATGTACTTCTACAGAGTTATTCTACATTCTACAAGTCAGTCTTGCTTGCTTTCGTTTCTGTGAGGAGCTA AATACAGAATGTTTTCAATGTCTCATAATGTTCTGTGCACACCCATGGGTGCCTAGCCAAGGATAGAAAC TTGTATCTGATAAGGAAAGAGGATGCTGCAGTGGCCACACTCTGAGATCACCTCCTGCCAAATTATACTA GAGTGTTTTACATAGTCTTGAAAACACTAGGTCAAATATGTTGTCATCCAGATATGAAAACTCTGTGCAA CAATTCAGAAATACAGTGGTCAGGACAACTGGTATGTAACACTTTAGTTCCAGCCCACATTAATCTCACA TCTTTTCTGTGGTTTCAATAAAGAGCACACATGTTTGGAACACCATCTCCACTTTGTATGCTTATCCTTA TGCATCAAACTTTTCCTGTAATTTCCTACCCCGCACAGGTTCCTGTCTTGATCTCTTTTTCTAATGAACA GCAATACAGAAGTGTTGGCCAAATGAACACTTTCACCCCAAGTTGCTTTGCCATTGTGTTTCATCACAGC ATTAGTAACTCCCAGTAAGGCTACTGGTTACCCTGCATCTTCTATGTAGATCTTTTGATGAAGTTCCAGG CTTGGGGTCTTTATCTGACTGTTATCCTGGTTAATTCATGTCCACCAATAGAATATTGTTCTCATTTTCA ATATCATATGAAGTTACCTGTTTATAGGATCTTCCTAAAGCAGAAAACTATATAAAATGTCTGCTTACTT TAAGAAAAGTATGTGTCCTTGGAGGTAAATGGAGGAAAGGGTGGAGGATGTTGAGTAATGGAAGACTGGC TAATACAGGAAGGTCACAGAATTGAATGATTTTAAAAATTTTAACTCCTCATCAATGAGGTACTTCAAGT TGAGGCAGGAGTCCTTGATGCACAGGTGACCCAAAGGCCCCAGCCTCTGTTTCTTGGTCTGCTCCAGTCT CCCGGCTCAGCACAGTTCACTGAGTGACAATCTTTAAGATAGTCTTTCAGGATACAGTTATCACAGTTTT CAAACTCTCTTCATTGAAACAATATAATGAGTCAGAGTCCAGTGTGCCATTAATAGGCCCTATGCAATAG GATTTTACTTCATCTGTGGAAAGAAACCACCATGGCCTCATCAAGGCCTCATTTGATGATTAGTGTAGAA AGCGTCCCCAGAGCTTTGCTGGGATGAAAACCCTGGCAAGATGTTTAGGGCACTCTCCATCTGACAGGAC AATTCTATCAAGGGTTGCTTCCATATAGAGAGAAATCTCCACATCTGCAGTATTTCCCGATTTATCAACA GTTCTTTACCCCGTGTCATATGCATAAAATCTTTTCTCTGTAGAATTTTCAGTACTAAAAACTAACAAGG ATGTTGGTCCTGATATCAGACATAATAAATAAATAAACATCAAATTATACCCTAAACATGTGGAGATGGT TTTCTGTTATGAATTTATTGGCTCTCTAAAAAGTGTTAATTAAGCCAGGCGTGATGGCACATGCCTTTAA TCCCAGCACTTGTGAGGCAGAGGCAGGCGAATTTCTGAGTTTGAGGCCAAACTGATCTACAGAGTGATTT CCAGGACAGCCAGGGTTACACAAAAAAACCCTGTCTCCAAAAAGTAACAAAAAGAAAGTGTTAATTAACA TCTACACTTTAAGATAATTTGGTGGCAAAGGTTAATAGTCTGAAATACTAGATATGAAAGTATGACACCT AAATTGCATGCAGAGAATAAAGCTGAATTCCAATGAGATTCATGCCTTGTGAATCCATTCCAATCTGATC TCAATCTTCAAACATGTTTGTGCATAATTTACATCTTAATCAGTTGCTGGAAATTAAAAGGACATAAGCT AAACTTAAAACTCTTTCTATTTAATGTGTCAGAATAGAATATGTGCACTGAAGTTTTCATGTGCATAGGA TATCATTGTTACAATGATGGTTCAAATACGCACCAGTAGCACAGGTGATGCTTGACATGAAAAAAACTGT GACCAGAGCCCAGACGTTGATGTTGGCTGAAAGAGCCTAGTGTCTCACATTTGATCAAGCAAAGTGTTAT TCAGAGAAGCAGTGCCAGATTTGGGATCCTTCTTAATGGATCAAGAACATGAGGAAGATTTGGAAGCCAG ATTGATGAGATTACTAGAATCCTGGATCTGAACACACAACACACAATAGTAAGTTGAACTTTTCCACGCA ATGCTAAAGATAAAGCATCCAGTATCATGTTATACAGGACCAAGGTGAGGATCAGTCTGTGGGTGAAGTA CAATGAACTGGAAATTGTTTGGATTTTAAATGTTGGTTTTTAAATTATATATAAAATATATATATATATA TATATATATACATATATATATATATATATATATATATATATATATCGGAACCAACATTTAAAATTTGGAT TTTAAATGTTGGTTTTTAAATTATATATAAATTATATATATATATATATATATATATATATATATATATA TATATATATATATATCGGAACCAACATTTAAATTCCTAATTCTTAAGGACTTCACTTTTGACCAAAAATA TTTGAAATGGGTTATTTAGGTCTGTGGTGGTTTGAATATGCATAGCCTATAGGAAGTGGAAGTATGGCCA TTTTGTAGTATGTGTAGCCTTGATGGAGGAAGTACATCACTATATGGTGTTGTGAGAGTTGAGATCCTAT GTTCAAGTTCTGCCAAAAACAGAAGAGAGTCCCCTCCTGGATATTTGAAACAGATGCAGAGGCCCAAAGC CAAACATTGGGTATATCTCAGGGTTTCTTATGGAAAAGTTGGCTGAAGGATTGAAGGCCTTAAAGGGGAT AGGAACTTCACAGGAAAACCAACAGATTCATCTAACCTAGACTCTAGGGGTCTTTCAAAGGTTGAGTCAC CATACAAAAAGGACACAATGGCTAGACCTAGGTCCCTGATACATATGTAGCAAATGTGCAGCTTAGACTC CATGTGGATTCCCCAACGACTAGAGCAGGTATTGTCCCTGAAGTTATTGCTTGTCTGTAGAATACATTTC CCAACATAATTGTCTTGTCTGGCCTCAGTAGGAGAGTATGTTCCAATCCTGGAGAGACTTGATATGCCAG GATTGAGAGATTAAGGGTCTCCCACCATCTCAAAGTTCATGGGTATGACAGGGGAGGGGCAGTGCAGGTT TTCCCCATCTCTGATTTCCAGCATTTGCACCCCCAATATTGAGGGGGAGGTCTAGCAATTGGAGGGGGGA CAAACAGTATGAGAAAGCTAACTTTAGTGGTGTTGTCACAGTGTGGGGTATTGTGGGTGGGAGTGGAGAT GAATCCTCCTCCTCCTCAGGAGGCTGTAGCAGGTGCTTGCCCTCTTTTGGGACAGTAGAGCTGTGGTCAG TACTGGCCTGTTCCTGACAAATTTATTCTGTGCTGTGACCATTTATTCAAGGGAGGAAGATTTAAGACTT GCTATGATAACCTGACGAATCTGGACAAACAAGAGACAGGAGGTAACACCTGAGTTACTCTTTTGCTTGC TAGTACAGAAGAGCACTATAATCCACAAATTGGGGTCCCACATATTCTTGAGTGGGATCCAAGGTGCCAT AATTTCAGCCACATTCCAAAGTCCCTCAAGGTCTGACAGTTTAGTCTTTAGGCTTCTCCTAACCAAATCA TATTTGGAGTTCTTTAACATAAAAGGGTAGTTTCCCCATATCACTGCCAGCAAAACAGGTTGAATTTGGA CAGACAAGCAAAACTATCACAAACAAGACAGACTATGTGTGCGATCCAAATGTCTTGTGATTAGAGGGGA AGTTCTGCAGCTTATTAGGCTGTGTCCTTATGCCCACAAATGTCTTCAGATAGGAAAGGGCCATTGGGTG CCAATCCTTAGTTCTTCTGTTTCCTGGGCTGTCAGTTTTTATGTGGAGAAGACATGAACCAAATGCAAAG TCATGTATCCTACAAGAACTGACATACACAGACAAACAAGGAATAACAATGAGCTCTCTCATTCACCTAA GGGGGTCTTATAACATAGAACAGAAAGGGGCCAACTTAGCAAGAATTGACCAAAAGGTGGTCACCTAGAC CACACCACAATGCAGGAAGGATATCAATGAAGGAAGAACAAAGGAAAGGCCAAATCAAGTTTGTTGGCAC ATGACATAAAACAGTGCTCAAAAAACAATAGATGCGAGCAAAATGAAGCATTGGAATCATATCGGGGTAC ATAGGGGTCACTCTTACCCCTGTTCAAGTGCCAGATGTTGTTTTAGCCTTAGGCTCCCTGAGTTTGTCTT TAGGGGAAATGGGTAAGAATACACAAAAATAATGTCACAGAATCTTAGAGGCAGATGAGAAGTCAAAGCA GACTCATTTATTGCTATAGAAAAGAATGCCTTTATACCTTCTGTCCATAACACCTCTGCCCATATATGCT CATCTCTGTGAGGCAATGGACTACCTGCAGACAGGCTGGTATCCAGTCAAGCTGAGGTCTGAACCACAAT GGTCGTAACTCACCTAAAGGACGTGGTAGGCATGACGCGATAGGCGTTCCCTCATGCTCCTGGAACTCCG GCCCCTGCCTAAGGTACCACCTCCCACAGCCCCCACAAGAGAAGCATGGTCAGTAGTCATGTAAGCAATG GCCCAAGCTTCTGACCTTCAGCCTAAACTCCTCCCCAGTTACCTAGCAACAGTGAAGACCATAAAATGGG ATGTTAGGCCCCCACCTCACTCTCTTACTCCTTTGTTCCTTTACCTCTCACTTCTCTCAATTCTCTCTCC TCTTCCCTTTCTTTGTCTTCTTCTTTCCTTGTCTCTCTGTCTTTCTCTTTCTCTCTAGCCTTCTCTGCCT CTCTCTCTCTCTCTGTCTCTCTGTCTCTCTCTTCTCTCTTTCCCTTGAATTTCTTTTTTTCTTTTTTCTT TTTTTGATGAGCACAGTATAAATAGTTTATTGCATTGTTTCTGAATGTGGCTGTATTCTCTATTGTTCTC TTTCTTATTTTTTTTCTTTTTTTGGTTGTTTTTGTTTTTATAGTCTCGCACAGAATTCTACATCTACCAA ATCCCCATTGTGTACTTTAGTTTTGTCTTGTATTCAAAGGTTCTTTCAGAAAGAATCTTAAAGATGAAAT TTGCCTTCCAACTTTTCAAAAATACATATAAAGATAGATGTTACATAGTTCACGGAATAGACACATATAC GGAGATGCAGCAATATCTCCTTTGTGCCTCTTGTATATTCATGTTATTTCCTCAACTAAAAATACATAGC CACAAAGACATACAATGAGCACTCCACTTAGGAAAATATGGCCATATTTTTTTACATTTTTATGATTTTA TGATTAGGTATATTCTTCATTTACATTTCCAATGCTACCCCAAAAGTCCCCCAACCCTTCCCTCACTCCC CTTCCCCACTCCCACTTGTTGGCCCTGGAGTTCCCCTGTACTGAGGCATATAAAGTTTGCAAGACCAATG GGCCTCTCTTTCCACTGATGGCCCACAAGGTCATCTTCTGATACATATGCAGCTAGATACATATACATAC ACGAGCTCCAGGGGGTACTGGTTAGTTCATATTGTTGTTCCACCTATAGGGTTGCAGATCCCTTCAGCTC CTTGGGTACTTTCTCTAGCTCCTCCATTGAGGGCCCTGTGATCCATCCAATAGCTGACTGTGAGCATCCA CTTCTGTGTTTGCTAGGCCCCATTATAGCCTTGCAAGACAGCTATATCAGGGTCCTTTCAGCAAAATCTT GCTAGTGTGTGCAATGGTGTCAGCATTTGGGGGCTGATTATGGCATGGATCCCCGGATATGGCAGTCTCT AGATGGTCCATCCTTTCATCTCAGCTCCAATCTTTGTCTCTGTAACTCTTTCCATGGGTGTTTTGTTCCC AATTCTAAGAAGGAGCAAAGTGTCCACACATTTGTATTCATTACTCTTGAGTTTCATGTGTTTCCCTTGC ATTTCTATAATAAACCATAAGGAGTCTCTGCTCTACCAAGACCCGCTGCACACTCTGGTCAGTGTTGGGA ACTTTTCCCCTATTCCCTCTCTCCTATAACTCCGGGGCTACAGGGTGTCTCCTTTGGGTCCCGGTTGGGA GCTGTCTCTTCTCAACCCCCTGACTCATGGGTCAGAGGCCTAAATCTCCACCCAAGGCTGTGTGAAAAGC ACCGGGTGGTCTCCCCATATCTCCCTGTCCAGAGCACAGGAACTCTGGCCGGACATGGCATATTTTTCCT CCCCAACTTCTTCCCCGGCCTCCTCAGGGCTGTCCCTTCATTTTGTTCCCCACACATCTCCACATGGCTG CCACCATCCAATTGGCTGCTGAGGTCACACACTCTGTCTTTGTTCTTCTCTGAGTCTCATGCAGGTTTTG TGTTAGTAAATGCAAAAGTGCCTGCTGTGCATGCATGAACCATTACAGGCTGCTAGGCAGAACATGCTGA CTTGCTCCTGGATTATAAATCCATCACTGTGATAGCAGGACATATCCAGAAGACATTATTTAACAACACT CCTCTTCGTGCTCAACCTCTCATGGTCTTTCTACCTCCTCTTTCTTAGTGTTCTCTGAGCCTACATGAAA TGTGAACAAACCTGAAAAAAATCTCTGTGTCAAATACATAGTGTGGATTGGGAAGGAAAAACCCTGCACT TGTGTGTGGGCAGGGAAATGCTGGTGCTTTAACAGGACAAGTGCGGTGTGGCTAGAGAAAGTGATGAGAA GAAGGGAAGCATGAGAACTGGGTGGGTGGATCCCATGTCTACTGAAAATGCTATGTTGATGTCAAGATAT TGTGTCTTGTTACAAAAATTCATTGAGTACCTAGGTTATTCTCAGTATATAAAAGGGGAGGAATGAAACT AGAATTAAACACACACACACACACACACACACACACACACAAACACACACACACGAGTAGAGAAAAGAAA TAGTCATGAGTTGCCTAGTGAATGAAGAGCGGAAAATGGTGCAGTTAATAGGGATCATAGAAAGAACTGG GGGATGCACACGAATGGGTTTCCTGTAATTATGTCTTCATAGTAATCTCTGCTCAATAATCAGACAGTGA CAATGTATGCCTCATTTACAAGCCCTGATGGCCTCTCAGAGGAAAGCATCTCTCTGAAATGAATAAAGTT CAGAAATGTCCTAAGTGTCCCTGTCACAGGAAAGCAGTGTTGGTAACGTTTCCAGGAAGCTCAGTCTTTG TCAAATATCCACAACAAGAAGAAGCCATGTCTAGACAGACAACAGACTGGGAAAGACTGAGCTCATGTAT GGAAACACAACTATGTGTCATGCTTTTATTTTCACATTATACTGAGGGGATATGACATTATAGAAACACA GTTTGTTCTTTGCATATGCTGAGAGAGGAACTAGATGAAGTGTGCTAATTCTTAGCAATAAAAAGATATA CAAAGTGGTACTTTACTGTTTCAATTTTTGTAACTGGCATCATTGTCTTTGATATTTTATATCTTCCTTG AGCACTGCCTTCTCTCAAGTGTCCAACTTCAGAGTATGCTATAGCAGGAAGACTACCAAATAAGATTAAT TTTTTTGTACCCATGAAAAAATTATGTGCCCTGACCCCTGTTCTCTGAAAGAGGAGCCAAGTTCTGGATT CCCAGATCCTCATATTCATTGATCAGCAGTGAACACAGATCATTCACCATGGACATGGGGCTCATCTGGA TTCTCCTTATTGTTTTTAAAAAAGGATTTCATTGGGAAAAGCTGCCTCATATTTCTATGACCAGGAGAGG AAGATACAGCTAGAGACACAAGCCCCACCATGTAAAATCTGTATGGTACTCTCGTTTTCATTTTACAACA TTTTGCTTTTGCATTGCAAAGGAGCAATAAGGGTTCAGCAATCAGCACTGTAACTGTACTTTTAAAATTT CCACCTCTACCCTCTGTGTCCTCTATCTAGTGATAGAAAGTGAAATAAGCACATAAAGCATGGGACAAGA AGAACACACAGAGGAGCAGAGACTGGCTACAAGTGGCAGCCAGCATGGAGCTGGACAGAATTTAAAAAGA TAAAGAGAAATGTCATGGCATGAAGCAGTGACTTTTTCTGTTCACAATGCCTGCAAAAACTTTATGGAGC CTACTACTGCTGAGATATGCAGGTTTGGTTCCAGAGAGGATTTTCTGTTTTATTTAATTTGCTTTTAAAT TTTTCTTCTTTGTATAGTCTGTATTTTTAAAAAGGGGTTGAATGAATACCTAAGCAGTTGGAAAATTTGT GAGTAGAAACCGAGGGCCTGAGAAACAAATAAATCAAAATTTTGAAATGGCAAAAGAAAGGGTTCATTCT ATTTTCTCTTTTCAGAAAAGGTTGCAAAGTTGGATGTGCAAATGGAGATGTTATGGGTGGACTTGGAACA TCTTGGGAAGGAAGGAACCCAGGAAAAGAAAGAATGAAATGGCTCAGCCTCAATGACTGAGCAAGAGAGT TAGAGTCACCCACAGGAGAAAAGAAGAAAATAAACAGCACTGGGGTGAATTTGGGTCAGAGAAAACCTTA AAATGCCAGAGGACAATGTGATGAAAGAGACCATTTGAAAGAAAGGGTTAACCTCATTCCATCAGTCATG CCAGAGTTAGAGAATGGGCAGTCTGGAGTTACAAGATCACGATTAGCCTTCCCAGTATGTATACAGCATA TTCCTGATAATGAGATGAAGCAAAGTTTGACAAGGTAAGATTGTATCCTATAGTAATATATAGATCAAAG GCATGTTGCAGTAAATCTCCAACAGAAATAAGCCCGGGCAATGAAAACACAACTCAATTAATATGAATAC GTGCTGTGCACCTAGACTGGGCAGATCTACCACTCCACTACCATATGAGAGATCCCTTATAACTTGTGGT TTCTCCAGGCCAGCTGCTTCTGCTCTACTTTCCTTCCTCCTCCTCCTATGTCATCCTCTCCCTCACTCTT TCTCTCCCAAAACTTTCAGCTGCACCTTCCCCTCTTCATCCCCCAATCAGTGGCTCTAGCCTTTATTTAT AAATTAAGGTAGGAAGAAGGTTTATAGGAAATTACCTGAGTGTTGACGTGTTGATAACCCCACACAAGAG AACAAAATTAATATCAAATATGATTAGCTCCAGGGTTATATGCAACAAAGGCATGATAAAGTGGTCATGA TTTCATATGAGATGTACTGGACTCATGTGAAAAAACGTTTAAATACATGGGCTTACTCAAAATATAGACT CATACCCCCAAGATGGGAAGAGACTGGTAACATTTGTACTGGAGGCTGGTCTGCTGTGGCAGCCATTAAC ACAGTGGAGGGAAGAGCCTCAAAGAGTGAAATAGAGGAAGGGGAACAAATACAGTAAACCACCAGTTACT ACTTAAAGGGCAGTGTTCTGATGTCCTAGGGAGGGCACTCCAAAATACCAATGGCCTTATCCTTAAATTT ATTGTTTAATGAGCCTACACTGATCTCAGTGGCCCACTACTAAGGAGAAATGACAGGCACTTGAACAGCT GATATGAGAACAGCTGGAGGCACAGTATACAGAAGAGTTTACAAGCCTATGGAACTCCCCTGTATTTTTT TATGATGAGGAAATCTGAAAAATGTAAAATGTTGAAAGATTTAAGAGCAGTTATTAGAGTAGATCAACCA ATGAGTCTCTTGCAGCCTGGAATTCTTTTATCTTGTTTGTAACAACAATCAGGCTCCATAAATTATAAAC TATAAAAATTTACTTTATAAATTATAAATATATATATATGTGTGTCTGTAGATAGATAGATAGATAAATA GATAGATAGATAGATAGATAGATAGATAGATATATGGATTTGATGGTTGCAGCAAATTCTAATTGGTTCC AACAACCCTACTCACATAATACATTCATACAGGTTTGATAGATGGAGCTAAGTTTTAAATAAATTATTTT TCTCCCATGGCTTATATATACACCACCAAAATTCTCAAAAATTAAAATGTGATTGTGTTGTATTTTTCTT TTACTGAATGACTATAAAAAGTTACAACATTCTCCATAAATTTACATGAAAAAATATTATGTAGTGCAGG TAAAGAAAACAAATTGACCCAAGAATGGTGTATATTCATTACTAAGCAACTTTTTAGATTCACAAAGTGT GGGTAAACAAGGTAATTTTTTCAATCAGTTTTTTTTAACTGGCAGGCAGCAATTCAGAGTTACAATGAGA AGATTAATAATTTTATTGTGTATTTTAAAATAAATCTTACAAAAATATTAATAGAATCACAAATTTATAC CTTTGTATAAAAACAATCAGTCATTTCTACTTTAAGAAACAGAACTCACATCTACTCATCAATCATTTTA TTAAGTCATATTACAAAGCTGAGTGCTAACATGGGTATAAGAAAACCATAACCTTATTCACCAGCCCAGC GTCAAAAAGAAAAAAACCAGTCATATCAGCTGCTGCTTCAAGAGTTCTTGTTCCTTGACATTAACAAAAT CCCTAGCTTAACTATTAAATTTTTTTTCAAAACTTCTAATTGATCCCTTAGATAAATGTTTGTGCTAACC ATCGGGACACATCCCATGAGTTCTGAAGCAGTGTGTTGTTCTTCATGCATGGCCCTTTTGTAGAGCTGTG AGTGTAGGGGAAGAGGGGGGAGAGAGAGCCCGTGTCCAGCCAGAGATCCTGTGCTCTGGGCAGGCAGACA CGGGAGGACAACGGAACACTTTTCACTCGGCCTTTGGTGGGCATCTGGCTGTGTGAAGTTACTGACCCCA CATGGTGGGGGATGGACAAGGGGCAGCCCCTGGTACCAGGAGCCCCAGGGCTACACTCTCGGCCCCAGAT ATACAAGAAGAGGGCAGAGGGAGAGAGGCTCCCACACAGGCGAGAGTCCTTAGTCTGGTCTGTGGCTGGA GCAGGGGAATTCCTTCTGATTGGAGATTAGGCACAGCTGATTAGGGGAAAGCCTACCCCATCATCCAAGC ACAATGGACTTTGAGGAACAGAGCCAGTCTAAGCTTTTATAGCTTTATGGTAGAAAGGCAGGGAGAAAGG AGAGAATGTGGAGAGAGACAGAGAGAGACTGGCCATGGCCAAGAGGAGGGAAGGGGGAAGAGAGAGAAAC AGGAAAAGCTAGAAAGTAAGATAAGAGAAAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGCAAAGTG GGGCCAAGCAGCCCCTTTTCTAGTGAGCTTGTCATCTCACAGTTGCTAGGTAACTGGGGAGGAGTTTAGT CTGAAGGTCAGAAGCTTGGGACCTTGTGTATGTGACTACTAACCACAGCTTCTCCTGTCGGGACTGTGGG AGCAGTAACTTTGACAGGAGACAGGGATCCAGGAGACATGAGGGAACACCTTCTGTCCCACATAGGTGGA ATCGGTTGTACCAGGGTTCAGAACTCAACTCCACTGGAGACCAGCCTATGTGTGCATAGCCCATTGCCCT ACAGCCTTTGACATTCTACAGAGTTCTGAGACTGGTAGAGATACAAAATAGCTAGGATTACACAGAGAAA CACTGGCTTGACAATAAAACTTCCAAAACAAATATAAATGAATAAATACACAAATTGGAGCAAGTGAACT TGCACTAAATCCATCAAAACTTGCACAGGATGGCTGTCTCCATAATGTTCAGATCTACTTCTATTAATGT CATTAAGAAGACAAAGATTATGCACAAAAAGAAACAAAACAAGAAACCCAAAAGGTAAAGGCTCCAATTG TAAGTAATAGACCCACAGTCTCTGAGGCCTGACTGTGCAGGTCCTCACCTTTGGTAATCAGCACTGAATG CAGACCACTCCTCATGGACTTTGGGCTCAGGTTTGTTTTCCTTGTCCTTATTTTAAAAGGTAATTCATAG AAATGAGATCCTGCCAGTATTGTGTACATGAGAAATAGAAAAATTGGTTTTCTTTGCTCTATTTTGTTTT GTTTGGTTAATGACAGTTTCCAAATCAGCATTCTTTGCTTTGAGGTGCCCAGTGTGAGGTGAAGCTGGTA GGGTCAGGGCAGCCTGGAGGGTCCCTGAAACACTCCTGTGCAGCCTCTGTAGTCACTGTGAGTGACTACT GAATGACCTGGGTCCTTCAGGCTCTAAAGAAGGGGCTGGAGAGGGTGGAAATAATTTTTAATGGTGGAGG TAGCACCTATTATCCAGACACCATGAAGGGCTGATTCACCATCTACAGAGATGATGCCAGAAACACACTT TACCTGAAAATAAACAGTCTGAGGTCTGAGTACACAGCCATGTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGAGTGTGTATTCATATATATATATATAGTGTGTATGTATATTCATAT ATATATGTATATTCATAAGGTTGAGTAAATGGACTTAAACTGATTCCATCACAACTTCCATGGGATGGAT ATTTCCCAGTGTTAAGACCTGTCAACATCACTGTCATTCAGGAGACAATGATTATGCACAACAAAACAAG AAACCCAAAAGCAGAGGACTCCAATTACAATAGACCCAGACCCACAGTCTCTGAAGATTGACTGTACAGT TCAACCCAGCCCTGTACTTCTCTTCCCTAGAATTTACATTACTGAGTAACTGAGGAAAGCTCTACAATAT CTGTTCTCTATAGTGGTCAACACCTCCAAACACAGGTTACCCATATTCATGCCTGCCTTCTGCTACACTT CTTGCCATAATGTAGACTACTTCAGCCTATTTTGTACTCCAGTTAACGAAACTCAAGACTAGCTGCATGT TAGTCCTTATTCTGAAATATTATGAACAGGTGACCTCCCATCATTCACCAATGCAATAATCATATTTAGG AATATGAGGTTTTATGAGATATAAGCACAAGGGAGAGAAAGTAAGAAAACTACATAGATATATAGACTGA CATAAATCAAGACTTGCATGAGCTAGTGCCCAAGTACCATGTCCCTAAGTGGCAAGGAGTATCTTTTGAG CCTAGTGAGATGAGGTACAAATCGGACTCTTACATCTTTTTAGATAAACATGTGAGATCAATGGACTAAA GGCGTGAGCTGGGCTTCCTTGCAATCCATTTTCCATACAAGATAACAATAGATCTGGCTCCACAGACACG ATGAGAATAGTCTTAATTAGTTCTTTAAGTAGAATGACTGATCACTAAGAGCCCAATTCCATACTAAATA CTCTTCTGGATTATGCACAGATAAAAATTGCACATAGGGCATGGGGCACTGATCTCCCTGCACTACATGA ATGGGGGCTCATTTACTAAATGTTCCCATGTTTCTTCCTAGTGCTGCACAGAGCAAATCCTACAACTTCC TGCTTGTCTACAATGTAAACTCCAGACAGTACCAGAAAATCATTCCTTATGTTCCTCTCCAGGTGCTTCA ACAAGCACAGTGCAAATTTCTGTCACCCTG Vaximmutor Vaximmutor Ighg1 Mus musculus 16017 AC160982 Vaximmutor Ighv1-9 Mus musculus 668478 AC073561 10090 12 114583568 114583861 immunoglobulin heavy variable V1-9 Also known as Igg2a; Gm16697 >gi|372099098:114583568-114583861 Mus musculus strain C57BL/6J chromosome 12, GRCm38 C57BL/6J GTCTTGCACAGTAATAGATGGCAGAGTCCTCAGTTGTCAGGCTGCTGAGTTGCATGTAGGCTGTGTTGGA GGATGTATCTGCAGTGAATGTGGCCTTGCCCTTGAACTTCTCATTGTAGTTAGTACTACCACTTCCAGGT AAAATCTCTCCAATCCACTCAAGGCCATGTCCAGGCCTCTGCTTTACCCACTCTATCCAGTAGCCAGTGA ATGTGTAGCCAGTAGCCTTGCAGGAAAGCTTCACTGAGGCCCCAGGCTTCATCAGCTCAGCTCCAGACTG CTGCAGCTGAACCT Vaximmutor Il10 (interleukin 10) Mus musculus 16153 6754318 AL513351.1 NM_010548 NM_010548 MGI:96537; Ensembl:ENSMUSG00000016529; 10090 ? >gi|6754317|ref|NM_010548.1| Mus musculus interleukin 10 (Il10), mRNA GGGGGGGGGGATTTAGAGACTTGCTCTTGCACTACCAAAGCCACAAAGCAGCCTTGCAGAAAAGAGAGCT CCATCATGCCTGGCTCAGCACTGCTATGCTGCCTGCTCTTACTGACTGGCATGAGGATCAGCAGGGGCCA GTACAGCCGGGAAGACAATAACTGCACCCACTTCCCAGTCGGCCAGAGCCACATGCTCCTAGAGCTGCGG ACTGCCTTCAGCCAGGTGAAGACTTTCTTTCAAACAAAGGACCAGCTGGACAACATACTGCTAACCGACT CCTTAATGCAGGACTTTAAGGGTTACTTGGGTTGCCAAGCCTTATCGGAAATGATCCAGTTTTACCTGGT AGAAGTGATGCCCCAGGCAGAGAAGCATGGCCCAGAAATCAAGGAGCATTTGAATTCCCTGGGTGAGAAG CTGAAGACCCTCAGGATGCGGCTGAGGCGCTGTCATCGATTTCTCCCCTGTGAAAATAAGAGCAAGGCAG TGGAGCAGGTGAAGAGTGATTTTAATAAGCTCCAAGACCAAGGTGTCTACAAGGCCATGAATGAATTTGA CATCTTCATCAACTGCATAGAAGCATACATGATGATCAAAATGAAAAGCTAAAACACCTGCAGTGTGTAT TGAGTCTGCTGGACTCCAGGACCTAGACAGAGCTCTCTAAATCTGATCCAGGGATCTTAGCTAACGGAAA CAACTCCTTGGAAAACCTCGTTTGTACCTCTCTCCGAAATATTTATTACCTCTGATACCTCAGTTCCCAT TCTATTTATTCACTGAGCTTCTCTGTGAACTATTTAGAAAGAAGCCCAATATTATAATTTTACAGTATTT ATTATTTTTAACCTGTGTTTAAGCTGTTTCCATTGGGGACACTTTATAGTATTTAAAGGGAGATTATATT ATATGATGGGAGGGGTTCTTCCTTGGGAAGCAATTGAAGCTTCTATTCTAAGGCTGGCCACACTTGAGAG CTGCAGGGCCCTTTGCTATGGTGTCCTTTCAATTGCTCTCATCCCTGAGTTCAGAGCTCCTAAGAGAGTT GTGAAGAAACTCATGGGTCTTGGGAAGAGAAACCAGGGAGATCCTTTGATGATCATTCCTGCAGCAGCTC AGAGGGTTCCCCTACTGTCATCCCCCAGCCGCTTCATCCCTGAAAACTGTGGCCAGTTTGTTATTTATAA CCACCTAAAATTAGTTCTAATAGAACTCATTTTTAACTAGAAGTAATGCAATTCCTCTGGGAATGGTGTA TTGTTTGTCTGCCTTTGTAGCAGCATCTAATTTTGAATAAATGGATCTTATTCG >gi|6754318|ref|NP_034678.1| interleukin 10 [Mus musculus] MPGSALLCCLLLLTGMRISRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSL MQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVE QVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS IL-10 plays an important role in Th2 type immune response. Il12b Mus musculus 16160 6680397 RP23-388G23.1 AL607030 NP_032378 10090 11 44400062 44414016 + interleukin 12b 6.5 35952.67 335 Also known as p40; Il-12b; Il12p40; Il-12p40 >gi|372099099:44400062-44414016 Mus musculus strain C57BL/6J chromosome 11, GRCm38 C57BL/6J CAGAAGGAACAGTGGGTGTCCAGGCACATCAGACCAGGCAGCTCGCAGCAAAGCAAGGTAAGTTCTCTCC TCTTCCCTGTCGCTAACTCCCTGCATCTAGAGGCTGTCCAGATTCAGACTCCAGGGGACAGGCTACCCCT GAACCAGGCAGCGTGGGAGTGGGGTAAGTGGATTCTGGGAGCATCTCGGATGGCTTTCCCCGCTGGTGGA AACCAGGGGCTTTGACGACAGGGGCTTCAAATAGTCATTTAAAAAGTCAGCTAATTACAGATGTGATGGC GCACACCTGTAATCTCCAAATCTGGGAGGCAGGAAACAGAATGCACAGGAGTTTAAGGCCAGATTCAGCT GTGTAATGAGTTTGAGAGAGTCTCAAAAAATTGAGCTACTAATAATATGTGCCAAGCATTGTGGACAGAT GTGTATTTGATAGAAAGGTTTGCTTTGTCTATATAGACACATATACTATGTGCATTTTCTTAAGTATTAA TGCATATGTGTTAAGGAGTGTTGTTAAAATTCTGTTCTTGCCTTAAGAGTGAAACTTAAAAAAAAAAAAA GCCTTTCATGTACCCCTTCAAGCCTAAGCTCATCACTGAATGTCCTACCTGTGATGGCCTTTAGAATCAG CTATGATAATCAAAGTGGAGAGCTGGGTTTGGGGGTTCCTTTGGATTTTAACTAACTTTGCCACCTCTTC AAAACTGGAAAGCCCAAGGCATGTTCCAAACTATTTCCTAAAGGGTTCTAAAATCTGTGAGAAAGGGAAG GCTAATCTTCATGGGTTGGCTTCCTCTGCTTGCCCAGGTGAAATCATGAGCACCAGATGGGGCAGGGTGA TGGGGCAGGGTGACATCTTGCTTTCACCCAGGGATCACTGAGCCTCAGCTGGGGGCTCCGTTTGTTCAAG CAAGGCATTTCAAGATGAAGTTAGGTCACCCCCAAGAATTCATTTTGCTGAGATCTGAAATACTGTGATT ATTGCACCAACAATCCATGGACTCTCTCTAGGCTAGATTCTGGATATAAGACTAGACAGATATGACCTCT GTCACACACCTCGGTATGGGAAAGGGGGGACAGACATTAAAGACGTAATCAAATGATTGTCATTTCAGCG ATGCTGAAGGGGAAGAGTAGCTATTTGGGGTCCTGTTTTCATTTGGGGCTCAAGCTGGCACTTTCTGTGG ATCATTTCATGAACTCCTAACCTGACAGAGTTATTTTTAATCTAGGAGTACAGGCTTGACAAAGTCAACA CAACTTGTCAAGGTCACATAACTAACAAGTGGCAGACTGAGGTCTGTTTGGCTGTAGAACCAAGGCCCTT TGCCCGTGCCCTCCTTGCCATCCCTGACAGCCACGCCATCTGTACTGTGCATTGTTGTTTCTGGGCATTT TTATTTTTACATATCATTTCTCTTTTAAATTAAAGAATAACAGAATGAAACTTGCAAAAGGGATGTCCAC TCAGGCACCCGCTGGCAGCAATGTTTGGAAGCATTTGCATGCTCTAGGGAGCTAGAGTATCCAGCCCAGG GTTCCTGTGCTCTTTGCACAAGACTTTCAGGACTTAAGAAAACTAATGAAAACAAACAAACAAACAAACA AACAAAAAAGAATTGTTAAAACATAGCATATGGAAGAATTGTGGAGTGAGAGGGAAACAAAGCCCAGCAT CTGCCAATCAGCCTGAAAGCCAAAGCCTGGCTACATACTGCACACTTTTTTGCTTGTGCAGATTTGATTA AGAGGCGACCTGCCATCATCTCCACCCTCTGCTGGTCTTCTATGATCTAAGATGTAGGTGATGCCGAAAT GAAGATTGTTCCTTGGACCATTGCCAGGGCTCCCTTCAGCCTGAAATATGGTTCACGTGTGATGCTCAAG TGCATCTGAGGCTGACAGAGGAAATGCTTGGCATGTAGCCAAAGGGGAAATCCCTGTGTTGAGAGCATAT GAAGAGATCACCAGGAGATGCCAGGAATCTCTGCCTTTTTTCCCTGCCCAGAGTCCTACCATCTGGGTTT CACAGCCGTGTATAATTGCATCACCTTCTCTGGGCATTTCTCTCTACAGCCTGGCAGATTTTTATTTTTC AACCCAGTGTGCACAAAACAGAAAGAATTTCAATTTTTTTCCTTAGCTTTTTACTAGTGCAAGGTATTAT ACAATGTCATCTCACTGCATGCCCAATAGCTCTTGAAGATCACATAAGCCTTATGTCTTTTTATATGAAG GCAGTCGGGCTCAGAGGAACTAAACCAACACCAAACAGCTACTGAGTAGTAGGGCCAGAATCCAAGTCCA TGTCTGCTTGACTCCAAGCCTATGAATGTAGCAACCAGCACCATTTCAGCTTGTACTTGTCTTGACTATA TGATAATTTGGACCTAGACATGTCCTGACTCCTAGTCCTGTCTCTGTCATGAAAAAGAAATCATAGCAGG GCCCTCAAGAGGTTAGCAGTACTTCTGTTCTCTCAAAGGTCGTGATTTCTGACCAAGGCCTGTTTATTTT TAAGGCATTTGCCTATAACATTTAAGCCTTAATTCATATGGGTTAGACTGTGGTCTCTCAAATCTTGTTC CTGACGGGCGGTGGCACCCTCCAGGATAGATGAAAAGTATTTCTGAAGGGAATTAGTAATCTACACTGAT TATCTCAACCACATTTATGAGTGAGGTATAATCTTTACAGTCTAAAGGTTTGTGTTGCCCTGGTGTTAAT CTAGACTGGGTTACAATACAGGATGGTTACTGACTTATCTATTTCATGATAGGTTAATCAATATGGTACA GTCTTCTGCCAAAGAACACCTTGAATTTTAGGAGTTCTGGACTCTCAAACCTAGCTAGCACATTTGCCCT CACAATGTGTTCAATGGATCATAACATCAGCTTTCAGCACCTCCAAGACATGTTAAGTCAGACACTCTGT TCTGGGCTGAGACCCAACAGTCTCCGTGAACCCAGGTTATACACACACCCAAGCTTAAGAAATGCTGCAT TTTGTTGTGAGGGAGAGAATTATTTTAGAATTTGGAGTAATCTCTGCCTAGGTTTAAAAAAACCTAATCC ACTACAGGTCCTTGTCAGAACTTGAAAGTTTACCCTGTGTCACCTTTCCCACACCTAAGCTGGTCCTCTA GCCTGGGGCATCCCAGTGCCTACTGCACAGCAGGGTGATTCGGATGCCCAGTGACACTGTTCTCGTCCAT ACTGGCAGCATGGCAGTACTTTCCCATTCTGTTCAAAGCTCTTTGGGTATTATGTTAATATTACAATCAT AACTAAGGAAATATCTGATGACTGTGGACCTGGGAAGCCACGGTCATCTCTAAGTTTATGTTTCTGAACC TTAAAAAAAAATAAATAAGATGGGTGTGGTGGCTCTCGCCTTTAATCCCAGCACTCAGGAAGCAGAGGCA GGTGGATCCCTGTGAGTTTGGGGTCAGCCTTATCTACATAGAGTTCCAGGACAACGAAGGCTGAAAGTCT GTCTTAAGAAGAGGGAGGGAGGAGATGATTGTGGATATCAGGAAGACAGTTCAGCAGGTCACATTACTTC TGCACAAGCCTGGTGACCTGGGTTTGAACCCTGAACCCAGAGTGAAAGGAGACAGTCAACTCCCAAAAGT TGTCCTCTGACCTCCACACATGCACACATACAATAGTCATACATCAATATATTTTTAAAAAACAAAATGG TATCAGGTAGCTGGGCCCTATGATTATTTAAGCTCCAGGTAGCCTCTAGTTCTTTATTTGGAGATGTTTT TACCCAAATCTAGAAGAGACCTCCTATAATCCTGGTCCAACAGTGTGAAAAGAAAGGGGAAAGTGAGTAT TTGGAAGGCAGATAAGCTGTTGATCGGGTTAATTGGATAACAGTTGACATTCTCTCTCCCATTTCCCCGC CTTTTCCTTCTCCTCTGCTCCCCTTCTTCCTCCCTCCTGCCCCTCCTCCACACAGATGTGTCCTCAGAAG CTAACCATCTCCTGGTTTGCCATCGTTTTGCTGGTGTCTCCACTCATGGCCATGTGGGAGCTGGAGAAAG ACGGTAAGCAGCCTTTTCCCTGTTTTCTTTGGAGCGCCAGCACCTAAACTTGATCTCCAGGCAGCCTGCT GGGAAGGGATAGTGAAGTCTGAAGTAGGCAACCGTGCATGGGATTGTCTCAGGAAAAATCCTCAGGGAAG ATGGAGGGTTAGCGTCTCTTCCTCCACCTATCTCTCTCCCTTGTTTGGTTTTCATGTTGGATGACAGGAA AGGAGTTTCAGGTGAACTATGGTGGTGTGTGGGATATGATCTTGGCCTTAGATACCAGAAAATGACTCTT CTAAACAATTGAAATTCCCTAATAGAGAATTTGTAACGTAGCAAGATTGTCTTCTACCCTCTCTTGTCTT GCATATTACAACGTGCTCTCAGTGGGCACCATGATGGTTGTCCTCTGGCTATTTTGGGATTGGCATTCAC ACTGACTTGGTTTCAGGTCATGGCTCTGCCAGGTATTGATACTGAACCTTTGAGGCTGGGTAGTCTTGTG ACCTCAGTTTTCTTTCCTTTTTTTTTCCCCCAGACAGGGTTTCTCTATATAGCCTTGACTTGTCCTGAAA CTCACTCTGTAGACCAGGCTGGCCTTAAACTCAGAAATCTGCCTGCCTCTGCCTCCCGAGTGCTGAGATT AAAGGAATGCACCACCACTGCCTGGCTGACCTCAGTTTTCTTAATTGGGTGTTCAGTATAGCACAGCCCA CATGGACTATTGAATATGTTAATAAATACAGAAAATAACATATGGTAGAGTGAGCAAGCACTATTATTTA AAACAAAAAGTCTCTGAACTTTTCATTATCCCAATGGCAATAAGATTATTAGTAGACAAATAATGAAGGC ATAGAAGGGGGTGCTCTCTGTCCTCTGTCCTTGTGCCATGTCACAGTTAGCAAATGTCCATCAACTCAGT CCTCAGATGGTCGTCACAGGATCTCAAATGTTTCATGGAATACAAGGTAATCAAGTCAGGAATTTCTGCT TTTATAGCTACACTTTAGTAATAATTAATATTTATTTAACACTCAATTTCGCAACAACCATGTAAAGTAG GAGCTATCTGTATTCCATTTTACAGATAGTAAAAACTGAGGTTCAAGAGGCTAAGTAAGTTTCCCACAAG GTCCAGAATAGAACTTGATCCCAGTTTAGTCTCACTTCAGAGCCTGTGTGCTTAAATGCTAGATTTTACA AGTAAGGCGTGAAGCAACTATTTCCTGTGGCCATAAAGAGGAAATGGGTGTAAATTATAAATGAAAAGAT TTCCCAGCTGCTGATTAGAAATAATCTTCTGAGAATCCAGGAAAAAAAAAAAAGGTGAGCCTTTTTTTCA TTCTGAAGAATTACAAGAGTTTGAACGATATTCGCAGGTAAAGGTGCTTGCCACCAAGTCCGATGACCTA GACTTCATCACTGCGATCAGCATGATGGAAGGAGAGGATCGAGTTCTCTAAATTGCCCTCTGCTCTCAAG ACATGAGCTGTGACACCTGTGCACACACACACACACACACACACACATGAAAGAATTAAATGCAACTTTA AAAAGAGTAATCTCCAGCCTGGCCACCCACGCCAACAGAATTTTGGATGCTTTCCTAAGTAAAGCCTTTG CCACAGCCAGATGGACAACTTAGGCCCTGGCAGAGTTAACCCCTTAGTTACTTCTGACCTGATAAACCAC AGAAATTTCAGAAAAGCAACAGGAGAAACACAGAGAGAGCTTGTGTTGGAGTCACAATGGTGGCCCGAAG CCCAGCAGATATCTAAGCTCTGATTCCAACGTGTCCAAGAAGTTCTTTCCCTGCCCTGGGGCTAAGGTCT TCTAGGGTTTCTGAGTTCAGGTTCTAAAAGGAAAATAATAGACAGCCTGTTTCATGGCCTAGGGGACAAA GGGAAACTGGGAGCCAAAGGGTCCTGTGCACTGGAATCAGCTCAGGCTAACGATTTCTGAAGAGCTTGGC CTCAAGTGATTTGTACCTCTTATCTTTGCTCCCCATTCAGCAGTGACAAATCCCACTGATGTAAAAGTAA GAGCTTAGCATTTCACAAGGCTTTAAGCTCCAATGCCAATCACAAAGCCACCATTCACAGGGCACTTAAG GAAACTGTCTTAAGCTTGCATACAAATTAGTTTAGATGTTACCTTCTAAAGAAAGCACTAATATTTTGCT TTCTTTTTTCTTTTTTTAAAAGAATGACATCAGACTAGGGCATGGCAGTTCATGCCTGCAATCCCTGAAC TTGATAGATCAAGACAAGAAGAAAGCCCAATTTTGTGGCCAGCCGGGGCTATAAAGTAAAATTCTAAGTA GCTGGCTGTCTCTTCCCACCCCCGAGGCCATGGCTCTCTGAAAGCCCTTTGCTGGCTCTGCTCCATAGCC GTGCTATTCCAAAACCCAGGCTCTTCACCACTATGCCTTCCATATTCAATCAGGCATCAGATGACTGGGG CCATCCTGATCTGACTCAGCAACCAAGAACAAACCTCACTGATGGGTTTTCAGGTGATGCTGATGATGGA GGTTGAAAGACGACCCTGCTTTACGGGTCACTGCATTAAGCTAGTCTTTCGATTAATAATTGATATGTAG GTAACCTTTGAAGCTTTGTCACATGAGGCTTTGGTGTGACCCACTCCAGAATACCTTATGGAGATATTCT GGAGGCTCCTTCAGAAACTAGCATCAGATATCCATTGCACAACACAGGAGACAAAAGATCCGAAAGACAC CATTTGTTACCCTTCACACTTGATGGGACCTCACTTAGCCCTTGGCCATCAGCAAAAACAGTTCCTTCAA AAACTGAGACAATCATCTACTGCTTTGTAAGAGTGTAAAATAGCTTTTAAAAACAAGTAGTTAATGGCAA TCTCCGTCCACAAATTTCATGCAGAAGAGACTTAAAAAAAAAAAAACCTAAATCTTTCTGGATAATTTTA AGAGTTTTTCAGTGCATTTGCCTGGCTCTTTGGCCTGTGCATGACAGTGATGATGATGATGGTGGTGGTG GTGGTGATGATGATGATTTTAAAAAGCTAGCACAGGGTTATCCTGCCGTAACCTTAGCTGTGCTAAGGTG CATGCTTTTTTACCATCTATCCTCACAGCTAATCCTATTAGCATAGACAATATTGTTCTTACCATTTTGT GAGGACTCTGGGGCTACTGGTGGTTCAGGACCTTGCTCAAAATCTCCTTTGCAATTAGAGAAGATAGGAT TTCTGCATCTTTCTTTCTCCACACCTATGCTTTAAGCCACTATGTTATTCTGCTCAATACTCTGCCCTGT GTATTCCCTGTGAATTACAACTGGAAGCTTACTCCCCCTCATCTCCATTCCCAACACACACACACACACA CACACACACACACACACACACACACACACTAGTGGCTTCTGACAAGGAATCTTGTGCTGTTTGCATCTTT TCTTGTTTCAAGGTCTTCCCTTCCTAATCAGAGACCGATTATTACTGGCCACCTGCCAGTTAAATGACTC GGTGGCAGCTATAGAACTGTTTCTCTGTTCTGTCCCCATGCCCTGAGATCTCTCAGCAAGCACTTGCCAA ACTCCTGTGAGCTATGACGTTCCAAGGGCCATGGGTGTGTCAAGACACTGAGTGAAACCCAGTTCCCGGG GTTTGTGAGGCTCCTGGTCCAGGTCCTCACTGCTGGTGCTCTCCAGTTTATGTTGTAGAGGTGGACTGGA CTCCCGATGCCCCTGGAGAAACAGTGAACCTCACCTGTGACACGCCTGAAGAAGATGACATCACCTGGAC CTCAGACCAGAGACATGGAGTCATAGGCTCTGGAAAGACCCTGACCATCACTGTCAAAGAGTTTCTAGAT GCTGGCCAGTACACCTGCCACAAAGGAGGCGAGACTCTGAGCCACTCACATCTGCTGCTCCACAAGAAGG AAAATGGAATTTGGTCCACTGAAATTTTAAAAAGTAATTCCATCCCCCTTGACAGTATCTTTTCTTCTAC CCCTCCTTAAAAAGTAATAATTGCAAGTGTGTGTGTGTGTGTGTGTGTGTGCGCGTGCGCGTGCGCGTGC ACTGAGGCTCAGAAATGTTTCTCATTTCCTTTGAAACAGGAACTCTCATTGGCCCATAGCTTACCAATAA GCTTTCTTGGTTAGCCAATAACATCCAGAGACCTTGTCTCAAAAAAAAAAAAAAAAAAATCTCACCCTTG TACCATGTAATGGCCAGTGATCCATCCACAGTAAAAAAGAAATAAATAAATAAAATAAAAATAAAAAATA ACAAAAACAAATTTCTTTATTAAAAGAAAACTCCTAAATATTTAATTTTTCTTTTCTTTTCAAATGCAGA TTTCAAAAACAAGACTTTCCTGAAGTGTGAAGCACCAAATTACTCCGGACGGTTCACGTGCTCATGGCTG GTGCAAAGAAACATGGACTTGAAGTTCAACATCAAGAGCAGTAGCAGGTAAGGGAGCTGCCATGATTTCT CAAGGTTTTGTGTGATCTACTTCATAACAAAAAGAAGTACAAACTGCCCATGGATTATCAGTGACTGGTA ATGGAACAGCAAAGTCAATTCTGTATTTTTTTTTTCAACTTCCCTCTGCTCGCCTAAGGAAATAGCATGG AGCTTTAAGCCAGCCCTCTGAGACTAGTTGGCTGGGTAGTTTGAATTCACTCATCCGAATTGCCTGGCCG TGCTTCATTAAGGCTCACAGCACCATGGGGAGGAATTAAAAAGAAGCCACAGCTACAAATAACTGGCCAG AGCTCGACCTTGTGTCAAAAGCAGAATTGAGTGCTCCAGGGCCTTGCTGTTGGAAGCCCTGGAACAGATT CCAGAACCCTCTGAGGCCTTTCGAGATTTTCCTAGAGCCTTTTCAACTTCTTCCCCCCCCCACCCCCACC CCCCAACCGTTATTCTGAAGTTCAGTTTCCCCAATGCACTTGGGCTTTTCCTAAAAACATCATCTCCCAG GGCTGCAGAGCGGGTGTGTCAGACTGCTCTACAGCTGACTTTAGTCAGCAACTAAATCCCTGAGCCACAT GGAGGGCAGGGAGTGAGCGCCCTGGCTCTCCCACTATTTAAATGAATAAACGGGGGACAGGGGGTGTGGG GGGGACGACTCCCTTTTCCATCTTTATCATGAATTTGCTATGACTTAACTTAGCTAAGGAAAATAATGGT TCAGGGTGACATCAGAGTCCTTGTTGCTTCTATGAGAATTTTCTGATCTGGTAAAGGTGGCCCCAAAGGA GACCAGTGTACAATGCAGATGCTTACATATAGAAACATGCCAGACCTCTATGTGCTTTAAAAAATTAGAC AAGCACGATACATTTTCCTGCTAGAGAAATCTTGCACGTGTTCCTGAAGAAACTAGTACATGGATAAGGG TCAGTGGAACATTTTGCATAATTAGCAAAAGCATGCTCAAATGGAACATCAATGTGCATCTATTGAAATA AATAACTCAACTTTCGTGTTCTGTTTAGAGAACTCCCCCACAATACTTTCAGAGAGGAAAAAGACGTTAC AAACTCATGCATATATATTTTTCATTTGTCTTTAAAGTTGTGCTACTCTTTGGATAAACTACATATGTAC ATATATGTCTGTGTGCATGTCTATCTTAAATATTCATTTGGAAAAACTCTAGAAGTAACAAACTGTGAAC AATGTCTATATGTGGGACTCAGATTTGAGTAAAAAGGAGAATTGTGGCGACACGCATGTTTTCTTTATAA TCATCTTTGTTGTTATAATTCTTTATAATTCAGCAGTTTCTCTTGCATTACGATATTTTCAGGTTCATTT AGGCAGAGATGCAACTTTCAGTCTCTTACAGCTAAGCTTCCACTCTGCAAAGGGCAAGGGGGTTTTTGTT TGGTTTTGGTTTTGGTTGTTTTATTCCACTGGAGAAATCCAACCAGCTGGACAGACTGGCCTCGGACTGT AGACACCAGGCACTCTATCTCTGATCTATAATACAGAACTCATTCTGTCTTTGCCATCAGTTCCCCTGAC TCTCGGGCAGTGACATGTGGAATGGCGTCTCTGTCTGCAGAGAAGGTCACACTGGACCAAAGGGACTATG AGAAGTATTCAGTGTCCTGCCAGGAGGATGTCACCTGCCCAACTGCCGAGGAGACCCTGCCCATTGAACT GGCGTTGGAAGCACGGCAGCAGAATAAATATGAGAACTACAGCACCAGCTTCTTCATCAGGGACATCAGT GAGTTTTGCCTGGGAGCAGGTGCTAGGGAAGCAAAGACGATGTCTGCCACGTGTCCATGATACCCTGGGG CATAGGATTGGCCTGCATTCTTTTCCATTCTCCTAAAAAGCATGATACAGACACTACCCTCTTCATCTTA CAGATGAGGAAATTAAAGGGTTAAGCTAACCTTTTTCCAGATCTCAAATCTCACAGACAGCAGACCAGAC CCAGACCTGCCCCAATGGTGGCACCTAATGGATTATGGGTTAACTACATGCTTAGTGTAAAGCAGAAACC AAGCACAGGGAGGGTTCCCTGATTGCTGAGCCCAGATACGAGACTCTATTAAGCAGAATGACTCAACCAG TAGGGACTTCCGGTCTGTGCAGTGCAGAAAGGGTTCTAAAATGTAGGATATGGCCAATAATGGTGACTTC CTCGAGTGGAAAGATCACTCTCAGCAGATCCCTCACAGGTGCTGCCCTGATGCTCAGAGAAGTGAAAGAG CTATTCCAGAGACTAGAAGGTGGAGGGCTCTGTCAGCCAAGCTCAGGAGCCTAAGTCCTAGCTCCAAAGC CTGGCCAGGATAATAAAGGATGCTTGTCTTCTTCTGCAGTCAAACCAGACCCGCCCAAGAACTTGCAGAT GAAGCCTTTGAAGAACTCACAGGTGGAGGTCAGCTGGGAGTACCCTGACTCCTGGAGCACTCCCCATTCC TACTTCTCCCTCAAGTTCTTTGTTCGAATCCAGCGCAAGAAAGAAAAGATGAAGGAGACAGAGGAGGGGT GTAACCAGGTAGGATTTATGCAGCCCATGTTGGGGCCATTGGAAAGATCCTGACAACCAGGTGCTTGCCT CAGGACCAGAACTGTGTTAAGGGCTCTCCATGTGAATCACACACAACCTATACAAAGTCCCAAAGGCAGC CAGTATTGCTGTCACCTCTCCACAGAGGCCAGGAAAGCTCAGAGAGGCTATGTTTTGCCCACGTTCTTCC AGCTAGAGCAGTGGCAGAACCAGTACCCAGCAATGGTTTGATGGAGAAAGAGATGGTTAGCTCAACCACT TCAATTCCTCCAGTATAGTCGATTGTCAATCCTCAACCAAAGTTGTTCATGATATAGAAACCTGGGATCC AGGAGCCTATAATGATTGTTTAAACTGAGTAAATATAGTAAATGCTAAAACATTGATGCTGGGACCCACC AAATAAGGATAATCTCTACTTCCTTAATATGAGCAATCATTTCACCTTACAAGATGTCCACCTTTACTGG GCTAATATGGTGACTTTCTACCTTTCGGGTCCTTAGCACAGTGATCTCTTGCTAGGTTTGAGGAACACTT TTATGAGCATTTAAGTCACAGAGCAAAGGATTCTTCCAACCTAGCATTGAGAATGTGATAGCAATTCAGC TCAGAGATTCAACCACTTCCTAAAAGCAGGAGCAAAAAATGGGATTATTTTGAAACAGATTTTAACTGCT ACCTAAGTTTCATCCCTGTGTGTATGTATTTAATATACCAATCTTAAATAGGTAATGTCTAGTGCTTTCT ACTTGCTAAAAGCTTTCTACGCATTTTTACATAAATGTTGTATTTAGCTGCTGCAATTCTTAGAGGCAGG CATCCTGGGACCTACTCATGAATAAGGAGAGACACAGAGAACCAGAGATATTTTCCTGAAGCACCACAGC TGTTTAGTAGCATTGCCAGGATTTGAAGTCACTTTTGTAAGCTGACAGGCATGGTAATTCTTGATGTCAA GACAGCTAAAGAAAGCTGGTCTCAGTCCCAACATCAGCTTCATATGGGAGTGGTTACCCATGGGACCTTG GGGTAATGCTGCTACTTCAAGAGGCAGAAGGGGGAGCAAGCCATTTCTGTTGGAGTTTACTAAATAGAAA AGTGAGAGTGGGGTGGTGGATGAATCATGCTGGAATATGAAGACCTGAATTTTAACCCCCAGAACCTCTG TAAAACTGAGTAGTAATGCCTGAACCTGGGCTCTGGAAAGAGGTGGGACACATACACACACACACACACA CACACACACACACACACACACACACACACACACCTCTGATTATAGCTTCTACCAGCAGGTGGTGCAAGGG GAGCGCCCATGCCAGCCTGCCTTATATTGCCTAAAGCCCTTACTTAAATTTTCCTCTCCTTTGCAGAAAG GTGCGTTCCTCGTAGAGAAGACATCTACCGAAGTCCAATGCAAAGGCGGGAATGTCTGCGTGCAAGCTCA GGATCGCTATTACAATTCCTCATGCAGCAAGTGGGCATGTGTTCCCTGCAGGTGTGTGAGCAGGACCGTG AAGCCCAACCACACCTGCACATTTAGTGGTCCTGGTGGAGGACAACAGGAGGCTTGTGAGGGAGGTGGGG GTGGAATTCCTGGCATAGGGATTCCAGTCAGAAACACATTTGTTCATCAGCAGATGTTAACTTGATGTGT ATGTTAGGTCAGGCACTATGCTAGGCCAAGGATATTACAATAAAGAAAACAAGCATGGGTCCTACCCTTG TACAGGGAAAGGCAAGAAGGACCAGAGGAGGAGGAAATCAGGAGTCAGAGTATGGAGAACTTGGAAGTAG ACAGAAGTTGGAACCATATCTTAGGGGTTCTGGGGAACCACTGAAGTCTTTTTAATAAGGAAATATCCCA ATCAAAGCCCTTTTAAGAAAAATCTCTCAAGAGAGTCCCTGGCAAGCATGAGACCAGCTGAAGTCCAAGC CCTGTAAGGAAGGGTTGGGCTGTCTCGCACAGAAATCCTCAAGACCCAGTGTCTGGGGATAATAATGTGT GGTGTGAAATAGGAGGGTATTGAGGCTATACCTCTGACCAATATGGACTTACCTACCCACATTCCTTTAC AGGGTCCGATCCTAGGATGCAACGTTGGAAAGGAAAGAAAAGTGGAAGACATTAAGGAAGAAAAATTTAA ACTCAGGATGGAAGAGTCCCCCAAAAGCTGTCTTCTGCTTGGTTGGCTTTTTCCAGTTTTCCTAAGTTCA TCATGACACCTTTGCTGATTTCTACATGTAAATGTTAAATGCCCGCAGAGCCAGGGAGCTAATGTATGCA TAGATATTCTAGCATTCCACTTGGCCTTATGCTGTTGAAATATTTAAGTAATTTATGTATTTATTAATTT ATTTCTGCATTTCACATTTGTATACCAAGATGTATTGAATATTTCATGTGCTCGTGGCCTGATCCACTGG GACCAGGCCCTATTATGCAAATTGTGAGCTTGTTATCTTCTTCAACAGCTCTTCAATCAGGGCTGCGTAG GTACATTAGCTTTTGTGACAACCAATAAGAACATAATATTCTGACACAAGCAGTGTTACATATTTGTGAC CAGTAAAGACATAGGTGGTATTTGGAGACATGAAGAAGCTGTAAAGTTGACTCTGAAGAGTTTAGCACTA GTTTCAACACCAAGAAAGACTTTTTAGAAGTGATATTGATAAGAAACCAGGGCCTTCTTTAGAAGGGTAC CTAAATTTAAAAGAATTTTGAAAGGCTGGGTATCGGTGGTATATGCTTTTAATTCCAGCACTCAGGAGAC CAAGGCAGGCAGATCTCTGTGAGTTTGAGGACAGCCTGGTGTACAGAGGGAGTTCCAGCACAGCCAGTGC CACACAGAAATTCTGTCTCAAAAAC >gi|6680397|ref|NP_032378.1| interleukin-12 subunit beta precursor [Mus musculus] MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIG SGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCS WLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELAL EARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEK MKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS Vaximmutor Il2 Mus musculus 16183 7110653 DN-144H19.3 AF195954 NP_032392 10090 3 37120712 37125953 - interleukin 2 4.63 18506.61 169 Also known as Il-2 >gi|372099107:37120712-37125953 Mus musculus strain C57BL/6J chromosome 3, GRCm38 C57BL/6J ATTTTTTTTTAGAGGAGAGCTTTATTTCTTGAAAACACTGATTAACATAGAGTTCACAGGAATAACTGAG AAGTTATTTTAGCGCTTACTTTGTGCTGTCCTAAAAATGACAGACATCTGAGCTTATTTATATTTGAATC ATCTAAATACTTTTATTAATGAGTCTACCTACACATGATATTTAACAATTCAATATAATAAATAATTTCA GATAAATAGTTTAAAACATTTTTGAGCCCTTGGGGCTTACAAAAAGAATCTTTAAAGATCCATATTTATC ATCTGAAGACTAGTAGTTACAAAAGATAGTAAACAATACATCCAAAAATAAATTAAAGTTAAATATTTAA ATAAATAGAGAGCCTTATGTGTTGTAAGCAGGAGGTACATAGTTATTGAGGGCTTGTTGAGATGATGCTT TGACAGAAGGCTATCCATCTCCTCAGAAAGTCCACCACAGTTGCTGACTCATCATCGAATTGGCACTCAA ATGTGTTGTCAGAGCCCTAAAAAAGAATATAAAATTGTAGGCTAAGGTAGCTTACTTTGCATATAATTAT TCTTCACGGAAGCTTTTAGGCATTTATTTTAACACTGGTTAAATATATTGAACATTCACATTTCTGAAAT GATCACAATTATTGGGTTATGTTTTATTATGATATCAAATGGTAAAGCATAAAAAATAAAGCTCTCCAGA GTTGATTACCAACGAGCATTTGATGGGAACACTGAACACTTCTGTAGAATTCAGTTGCTTGACCACTTTT TCTCTGGTAAGTGTACACAGCTATGCATCAATTTAGGCTTCAGGAATGGAGAAATGATACTTGTTATATG TTCTTTGCTAAATCTTAAGAGTCTATTATTCAATTCTTTCCCCCCCAAGTCAGAATGGGAACAACCCAGA GTAGGTTAGGACAAGCATCTATTGGGGGAAATTTGGAGAATTTTGTTATAGATATACCTACCCTGATACC AGTGTAGATGAATCACAAAGTTTGTACATGTATAAATATTATAAATTTGTTCTGTGGATTAGCTTTTTGA AAGCTAACCCTCTTTGCTAAAGGAGATATTTTAATTCTGTTTTTAGGTGACAAGCTGAGAAGGAAATATT TTGACAAATTTATGGGATGGCTTTTCCAAATCTGACTAATTCCTTGTAAGTCAAATGAGGCAGATTATTC CTTTTAAATAAATACTCCTTTTCAAATAAGCTGCTGTGGTCTTATATACCCTCTTTACATAGCTACTTCA ATATTCTAATGTCTTGAATTTTTCATTTTTAAAAATATGTGGCCCTTGAAACATTTTTCTAGTTCTCATG TAGCTCTAAAGTTTGTACCATTCATTAGGGGTGAACTTTCTGATGTTGGCCATAGATTTTAGAGCATCCA TCTTTGCATACGTAAGTACTCGATTTTAGAGCATCCATCTTAGCATACGTAAGTACTCACACCTTTCAAC GTCTAGATGATGTGTATTTCAGCCTGCTTTAAAAACCTCAAATGGAAGCTGGGCAGTGGTGGTGCAGGCC TTTAATCCCAGCACTTGGGAGGCAGAGGCAGGCGGATTTCTGAGTTCGAGGCCAGCCTGGTCTACAGAGT GAGTTCCAGGACAGCCAGGGCTACACAGAGAAATCCTGTCTCAAAAAAACAAACAAACAAACAAACAAGA AACAAACAAACAAAAAAAACAAAAACAAACAAAACAAAACCTCAAATGGAAACAGTGCACTGAAATGTGA CTTTGGTTTGGAAAGGACTAGCCCACACCCTCTTGGAGGCTTGCTGCTACTGTCTCACCAGAGTCCATGA TTCTTGTGCATTTAAAAACAAAGCTCTGCAGTGGGCTCTCCCCGTCCTGCTCTGTCCATTTTAATGGCTG CCATTTTTGGAGAGAAATGTCTGTTTTTCTACCAATACCAGCACAACTTCTCTGGAAAAACTTTTCAGAT AATTTTTTCTGATCTGATGAATGTAACACCAGCAAGAGTTGCTTGTTTCTTGTGGAATTCTACTCCGTGC TTTCTCTCACATCCAGTTCTATGCTGGTGTGGAGGGAGCAGAGTGTTCATGTTCCCAGTTTCCTTGCAGG TGATGGTAGGTGGAAATTCTAGCATCATCCTACAGTGGAAGGATTCACTTGCACAGTGACTTTAAACTTT GGCTGACTAAATGCACAGAACCCATCAAAGACCAGAAATGGCAAGCCACTTAAAAATGCATTTCTTCTCT ATTTTATTTCCAGATTAGCAAATAAAGCAACACCTTACCTTTAGTTTTACAACAGTTACTCTGATATTGC TGATGAAATTCTCAGCATCTTCCAATTGAAAGCTTTTGCTTTGAGTCAAATCCAGAACATGCCGCAGAGG TCCAAGTTCATCTTCTAGGCACTGAAGATCTTTCAATTCTGTGGCCTAGAGGAGTAATAAGCTTAACCAT CAGCTCAGCTCACCACATACTGAAGAGCCAGAAAGTTAGTCTGCAGTCTCTCTAATCAAGAGAAGGCAGC ACATAGCTTTTACCACTCCCTGTCCTCTGTAAAGGGGGACTGAGGTAATCGATGCCATAAATCTAGAAGA GCAAGAGCTCACAACCATAAGGGTCACCTTGACTGTTAGGCCACTCTAGTGAGCTCTTCTGGCTTCATTA GACTTTGTAAAAAGTCTGTGTTTCTCTACCAATGCATAGCACAAGTTCAGACTATTGTTCCAATCTACAA AGAAATCTGCAAGGTTCACATTCTAATATCTAATCGCAGAGTTGAGAATCACAGAAGGGTAAGGGACAGG AATCCTTGGATGCCAACTGTCATAGGCCTAAATCTTACAGATTAGGTTATCATGGCCCTGACTGAGGGGT GTCAAGATAGCCAGGAAGACACATGTAATATTTTTTAAATTTATACTTCTCTGCAATTACTAAAGATGAG TTTTTTAAAAACCCATATGTCAAGCTAGGAAGATGGCTTTTAAGGTAAAGGTACCTGCCACCAAACTTGA TGATTTGAGTTCAATTGCAGTGACCCACCCACATTGTGGAAAGAATGGCCCAACTTTCATATTTCGTCTT CTGACCTCCACAGTTACACTGTGATACACATGCACCTCCACACATACATACATACATACATACATACATA CATACATACATACATACATACATACATAAAATGTAAAATCTATTTGTCCCAATGAAAAAAGTCATAACAT TTTGGCAGAACACTTGGGAGAATTTTTCTGATCCATGTATGAAATAATGTTTAAATACATCATGCATACT TACATACATATACAGGGAAAAAGGATATTGTCTAGCTCTATAGTACACTATGATTTCCATAAAGTCAGCT GCTTTAAGTCTACAGTGAACTGTGTTATCTCCATTTTACCAAGTCTCACAATCAGTGTCTTTCCCACAAC CACATGAACGGGGAGCAAGGGTGGCCCGGTCGGTCGGTCGCTCCTTTCATTAACCACAGCCTTTGAATTA CATGCTTCTATTGTTTTTAAAATCTTACATACCGAATGCAGGTTAAATGAAAACATCAGCAGAATTTACC ACATTACTCTTGTTTTCAATAATTATTTTAAAACTTAGATTTCTAGGAATTTCACCACTCTCTAAATTAA CATGATCTCTGTTGTTTCTCTAACAATCCTTAGAGGAAGAGGTTTGACAAGATAACACGTAGTTCCTGAT TGGTCTGCTATTGACCTTCCAACTTGTTATGGTGTGTCTTGGGTATTCAGTGTGGAGCTCCTTCCCTCAC AGAACTCTCCAGTTTGTTACTGTGGGAGCTGAACATTATGTGACTGTAATTAAGCTGGAAGAGCAACTGG GGCCTTTCTTTTTCATCTCCTGTAGAACAGCTAGAGGCAAAGTTCCCCTGACTCAATAGGAATGGGTCAA CAGCTGCCTCTTTGTCCCCTTAGATCCCATTTTGCTCTAGGCTTGGATTTACTTGGGACAAGCTCTTTCT AGGACCAAAGTTATCCATTCTCTTTTGACTTTCGGGACTAGAGCAAATTTCTACCCTTGCTGGAGTTCTA AGAGCTTGTGTAAGCTGTTTTGTATCACACAACATTCTGCCTCCAAAGGAGACCAAAAAACCTTTCAATA TTGAGAGATAGTATCATGTAGTAATATTACAGCTCTCTAGTTCCACTAGTTAAAAGAGACTGTATAAATC CAAAATTGTTTTATACATCTCGGACTTCTTCTCTAGCACTAATAGTGTGTTTATGTGCTGTTGGCCAGAC ACTGTTCTGAAACCTGACAAGTGATAAGCATAGCAATGATCATTTATGTAAGTGGGTGAATGAGCATAGA CAACTTCCTGAGGCCATGAGAGCTAGGCCCTAGCAGGGCTGGACTCCACTGCAGATCATTGGCCCTAGAA TCTTAACATTGGTTATGCTGCTCCTCAAAAATGCAGTCATAGGAATCATTCTTAAAACAGTAAAGTGTGT GTAAAATGAGACCCTCTCAAGTCAATTTTTACATTTAAGAAGCTGAAATAATGCACCTCTCTCAAATATC TGTAGGACAAGCATAAAACAGAAATCAACAGTCTTTAAATTATCCCAGCATCAAAATGCAATCATCTTGC ATTTTCTTCTCATTCCCTTTCCCAACACATAAATACACAGGAAAAATGTAAAGAAAACCATTTAAAAGAA GTACCATTGTCAAGATTTTTTTACAGAGAGATATCAGAGAAACTTATTTTCTTCAGATGAGGTACTATTC ATCTGGTCACATGTTATTTACACGTTATACACATGTATGTTACTTTGAATTCATTAGAGCACCAGTTAAA CACAGAAACTCACTCACCTGCTTGGGCAAGTAAAATTTGAAGGTGAGCATCCTGGGGAGTTTCAGGTTCC TGTAATTCTGAGAAAGCGTAACACATTATTATTAAAGGTTATTAAACACAGCCTTTGGCAAGAAAGCTAA AGGTATTGCCTATAGATGGGATGGCTGTGCACTTACCTCCATCCTGCTCAGGAGCTCCTGTAGGTCCATC AACAGCTGCTCCAGGTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGTGCTTCCGCTGTAGAGC TTGAAGTGGAGCTTGAAGTGGGTGCGCTGTTGACAAGGAGCACAAGTGTCAATGTGACACAGGATGCGAG CTGCATGCTGTACATGCCTGCAGGACTTGAGGTCACTGTGAGGAGTGATTAGCAAGGGTGAT >gi|7110653|ref|NP_032392.1| interleukin-2 precursor [Mus musculus] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKL PRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFEC QFDDESATVVDFLRRWIAFCQSIISTSPQ Vaximmutor Il4 (interleukin 4) Mus musculus 16189 10946584 RP23-188H3.4 NM_021283.1 NM_021283.1 10090 ? IL-4, Interleukin 4 >gi|10946583|ref|NM_021283.1| Mus musculus interleukin 4 (Il4), mRNA GGATCCCCGGGCAGAGCTGGGGGGGGATTTGTTAGCATCTCTTGATAAACTTAATTGTCTCTCGTCACTG ACGGCACAGAGCTATTGATGGGTCTCAACCCCCAGCTAGTTGTCATCCTGCTCTTCTTTCTCGAATGTAC CAGGAGCCATATCCACGGATGCGACAAAAATCACTTGAGAGAGATCATCGGCATTTTGAACGAGGTCACA GGAGAAGGGACGCCATGCACGGAGATGGATGTGCCAAACGTCCTCACAGCAACGAAGAACACCACAGAGA GTGAGCTCGTCTGTAGGGCTTCCAAGGTGCTTCGCATATTTTATTTAAAACATGGGAAAACTCCATGCTT GAAGAAGAACTCTAGTGTTCTCATGGAGCTGCAGAGACTCTTTCGGGCTTTTCGATGCCTGGATTCATCG ATAAGCTGCACCATGAATGAGTCCAAGTCCACATCACTGAAAGACTTCCTGGAAAGCCTAAAGAGCATCA TGCAAATGGATTACTCGTAGTACTGAGCCACCATGCTTTAACTTATGAATTTTTAATGGTTTTATTTTAA TATTTATATATTTATAATTCATAAAATAAAATATTTGTATAATGT >gi|10946584|ref|NP_067258.1| interleukin 4 [Mus musculus] MGLNPQLVVILLFFLECTRSHIHGCDKNHLREIIGILNEVTGEGTPCTEMDVPNVLTATKNTTESELVCR ASKVLRIFYLKHGKTPCLKKNSSVLMELQRLFRAFRCLDSSISCTMNESKSTSLKDFLESLKSIMQMDYS Vaximmutor IL-4 plays an important role in Th2 immune response. Il5 Mus musculus 16191 6754336 RP23-239O19.2 AC084392 NP_034688 10090 11 53720793 53725102 + interleukin 5 8.9 14616.09 133 Also known as Il-5 >gi|372099099:53720793-53725102 Mus musculus strain C57BL/6J chromosome 11, GRCm38 C57BL/6J GCGCTCTTCCTTTGCTGAAGGCCAGCGCTGAAGACTTCAGAGTCATGAGAAGGATGCTTCTGCACTTGAG TGTTCTGACTCTCAGCTGTGTCTGGGCCACTGCCATGGAGATTCCCATGAGCACAGTGGTGAAAGAGACC TTGACACAGCTGTCCGCTCACCGAGCTCTGTTGACAAGCAATGAGGTAAAGTATAACTTATTCCTTCAGC TTTGTTTTTAAGATCAGGACCTTGCTATACCGCTCTGACTGGCCTCAAACTTGCTATGTAGGGTAGGCTG TCCTAACCCCTACCAGATCTCCTTACCTATGTCTCCCAAATACTAGGATTACAGACACATTACCTTGCCT GACGCTATGGTTCTTCAGAATGCATAAATAGCTGCATTTGGCCTTTAATCCCAGAACTTGGGAGGCAGGG TCAGGTGGATCTCTGTGAGTTCAAGGCCAGACTTGTCTACGTGGCCAGTTACAGGACAGCCAGAGCTAAA GCAAGACCCTGATTCAAAATAATTTTTTTTCAAAACAAAAAAAAAAAACCCAAACCATTTGTGGCAATTC ATTTCTAAACATAAAGATCTGCTTTAAATAGTGCAATTATGGCTTGTTCCCTTGCCTTCTTGCTCCCGTT CTGTCCTCTTGTCCCACTCTCTCCCCATTCCACCCCCACCATGTGCTCATGGCCCGCATCTCTACTTCTC TACTCTCTTTCTCTCCCTCTCCCCTCCTTCTTCCTTTCCCTCTCTCTCTCCCTCTTCTTCTCCTCCTCTC TTTCTCTCTCTCTCCCTCTCTCTCTCTCTTTCTCTCTCTCTCTGCTTTTTTCTATCTCTACTACCCTCTC AACTCCCCTCTCCATGCCCTGAATAAGCTCTATTCTATACTAAAAAAAAAAAGTGCAATTATGAATGTGT TAGTGTTAATGCACAGGTGATAACCCTATCACCAGCAAGCATTGCATTAAAAAAGGCAACGGACTCTCTT TAGGATGACCCTATGATGTTCTTTCCTTTGCAGACGATGAGGCTTCCTGTCCCTACTCATAAAAATGTAA GTTATTCTTTACTGCCGTGCTTGCATGAGTAAGTCAGCTTCGCATACTAAGCTATAAGTCATCTGCATCT AGCTTTCTGGTGTTGTGTGTGTCTGGGATGGGGACCTCTCTAGGTCTCAAGCTCCTGGGTTCAAGTGATT CTCTTGCCTTGATAGAGCAGCTGGGACACAGGCCTGTGCCACCACACCCAGCAGAGCTTTTGATTTCAGT TAAACTGTTTGACTTTCTTGGAAAAGAAAATTTATGTAGGTAGATATGAAAGTTTGTGCTTATAAATAAA AAGAATATGAGAGTGGCAAATTATGTAATCCCAGTACTTGGGAGCCAAAGGCAGGGGTAGTCTGAGTCTA GGGCCAGCTTAGATACATTGCCCTGTATGTATCAAAAGTAAATCCTATAAATAAATAAACAAAAACATTA GAGGGCTGGAGATATAAGCTCTGTTGATAGATGGCCTAATATGCTGGGTTGACTCTTAGCACCCCATAAA CTAAACATGGAAGTACCTGGCTGTAATCTCATGATGGTGAAATGGAGGCGGGAAGATCATAGGTTCAAGG TCATCCTCAGCTACATTTTTGAGCTAGAGGCCAGCCTGGGCTATGAGACACGCAAAAACCACCAGCCAAT TAATATTAGGAATGGCTTTGAGCTAGATCTGTTATGTAAGTGGCCAGCTGGAGCTGTCAGTCATACATCT CACAGCCTCACAAGATTCTTTGCATGGCGAGAGGTCCTGCTGGGCTCCCTTTGGCTCTGTCCATGGCTCT CTTCATCCTAGTGCCTCTCTTTGTTTTCCTTGTCTTATTTCTTACTGCTGAGGATCAAGCCCAGGGCCTT CAGTGTGTGAAGTGAGCACTCTACCACTGAATTCCAGAGCCCGCCCACTCTAATGCCTTTCTGAAAGTAT TAAGAGTTTAGGGTTATATATTCCTTTTGTTTATTTTATGTGTATGAGCATTTTGCCTGCATATATATAT ATATATATATATATATATGTGTGTGTGTGTGTGTGTGTGTGTGTATATATATATGTATGTATGTATGTAT GTATGTATGTATGTATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTCCACGTATGTGTCTATGTG TCTGGTGTTCCTGAAGGCTAAAAGAAGGGCATCAGATCACCTGGGGCTGGATATGCAGATGGTTGTGAGC CAACCATCTGGATGCTGGGAACTGCATCAAGTGTTCTTAACCACTGAGCCATCTCTCCCGCTCAGAGGGT TATATTCTTAGGTAATGATAGAAAGACATAAAAATATCATGAATGCCTTTATTAATAATTTCTAAACAGT TTAATGAATATGACTATGTAGTGATATTGTATACATTTCAATATTATCTTATTCTAGCGTAAAGTACATT ATTTAACTTTTTCTAAATAGAAGAAAATTCATCAGCCTAAATTTCAAAAGAAAATATTAATATGGGTGTG GTACCACTCACCTTTAATCCAGATGGTTGTGAGCCACCACAAGGGTGCTGGTAACTGAACCCAGGTCCTC TGGAAGAGGACCCAGTGATCTTAACCACTGAGCCATCTCCCCAGCCCCAATCCTAACTTTGGGTTCATTT TTTTGAAATGATCTCATGTAGCACTAGCTGGCCTCAAACTCTATGTATCAGAGGCTGGCCTTCAACTCCT GATCCTCTTACCTCAACTTCCTGAATGCTGGCATTACAGATAAGCACCATCACATCTTGTATTGTCTGGG GTTTTTTATTGATGCATTTAAATTGCATGTATTTATTGCATATGGCATGATATTTCAAAATATGTGTACG TTGTGGGCAGTCTGATCTATTTGCTTCTTGATAATCTTCTTTCAGCACCAGCTATGCATTGGAGAAATCT TTCAGGGGCTAGACATACTGAAGAATCAAACTGTCCGTGGGGGTACTGTGGAAATGCTATTCCAAAACCT GTCATTAATAAAGAAATACATTGACCGCCAAAAAGTAAGTTCCCCAGGGACCCTGTGAATCCGGCTGCAG CTGGTTCTCCAGGAGCCAACCTGACAGTCTGTTCTTTTCACAGGAGAAGTGTGGCGAGGAGAGACGGAGG ACGAGGCAGTTCCTGGATTACCTGCAAGAGTTCCTTGGTGTGATGAGTACAGAGTGGGCAATGGAAGGCT GAGGCTGAGCTGCTCCATGGTGACAGGACTTCACAATTTAAGTTAAATTGTCAACAGATGCAAAAACCCC ACAAAACTGTGCAAATGCAAGGGATACCATATGCTGTTTCCATTTATATTTATGTCCTGTAGTCAGTTAA ACCTATCTATGTCCATATATGCAAAGTGTTTAACCTTTTTGTATACGCATAAAAGAAATTCCTGTAGCGC AGGCTGGCCTCAAACTGGTAATGTAGCCAAGGATAACCTTGAATTTCTGATCCTCCTGCCTCCTCTTCCT GAAGGCTGAGGTTACAGACATGCACCATTGCCACTAGTTCATGAAGTGCTGGAGATGGAACCCAAGGCTT TGTGCATGTTACCAACTGAGTTATACTCCCTCCCCCTCATCCTCTTCGTTGCATCAGGGTCTCAAGTATT CCAGGCTGACTTTGAACTCAGTGTGTAGCCAAGGGTGACCCTGAACTCTTGGTCCAGATGGACGCAGGAG GATCACATACCCAACCTTAGCATCCTTTCTCCTAGCCCCTTTAGATAGATGATACTTAATGACTCTCTTG CTGAGGGATGCCACACCGGGGCTTCCTGCTCCTATCTAACTTCAATTTAATACCCACTAGTCAATCTCTC CTCAACTCCCTGCTACTCTCCCCAAACTCTAGTAAGCCCACTTCTATTTCTTGGGGAGAGAGAAGGTTGA CTTTTCTTATGTCCTATGTATGAATCAGACTGTGCCATGACTGTGCCTCTGTGCCTGGAGCAGCTGGATT TTGGAAAAGAAAAGGGACATCTCCTTGCAGTGTGAATGAGAGCCAGCCACATGCTGGGCCTTACTTCTCC GTGTAACTGAACTTAAGAAGCAAAGTAAATACCACAACCTTACTACCCCATGCCAACAGAAAGCATAAAA TGGTTGGGATGTTATTCAGGTATCAGGGTCACTGGAGAAGCCTCCCCCAGTTTACTCCAGGAAAAACAGA TGTATGCTTTTATTTAATTCTGTAAGATGTTCATATTATTTATGATGGATTCAGTAAGTTAATATTTATT ACAACGTATATAATATTCTAATAAAGCAGAAGGGACAACT >gi|6754336|ref|NP_034688.1| interleukin-5 precursor [Mus musculus] MRRMLLHLSVLTLSCVWATAMEIPMSTVVKETLTQLSAHRALLTSNETMRLPVPTHKNHQLCIGEIFQGL DILKNQTVRGGTVEMLFQNLSLIKKYIDRQKEKCGEERRRTRQFLDYLQEFLGVMSTEWAMEG L7/L12 Brucella abortus RB51-AHVLA 479804332 CDD:100102 1198700 ? 50S ribosomal protein L7/L12 4.62 12562.32 198 vaccine strain >ENT73302.1 50S ribosomal protein L7/L12 [Brucella abortus RB51-AHVLA] MADLAKIVEDLSALTVLEAAELSKLLEEKWGVSAAAPVAVAAAGGAAPAAAAEEKTEFDVVLADGGANKI NVIKEVRALTGLGLKEAKDLVEGAPKAVKEGASKDEAEKIKAQLEAAGAKVELK Protective antigen manA Brucella melitensis bv. 1 str. 16M 260563601 BAB1_0562 CDD:32765 CDD:29324 224914 I 550431 551708 ? ManA family protein 390 N-acyl-D-glucosamine 2-epimerase [Carbohydrate transport and metabolism] >gi|82698932:550431-551708 Brucella melitensis biovar Abortus 2308 chromosome chromosome I, complete sequence AATGAATGATGTGATACGTAAACTTCAGGTGGAAGTGGAGCGCTTATCGTCGTGGCTCCACAACGACGCT TTGCCTCTTTGGTTGTCAGCCGGTGTGGATGCGCCGAATAGCGGTTTCTTTGAACGTATTGGACAGGACG GCGTCGCAACGGATGCGGATAATCGTCGTTCTCGCGTGCATCCGAGACAGATCTACTGTTTCGCCAAGGC TGGCGCCATGGGCTGGCAAGGCGAATGGAAGCAGACGGTTGAAGCCGGAATAGAATATTATGATCGCGTC TATCGGCGAGAAGATGGTTTCTATGGCAGTCTTGCCGGTCAAAGTGGCAAGATGATTGATCACACGTTTG ATCTCTACAATCAGGCTTTTTCCGTTTTTGCGGCCGCCCAGATCGCGGTTTCCATACCGGATCGGTTTGA TGAAATGCGTCAGCGAGCGCTTGGTTTGATGAATACGCTCGTGGCAGATTATCACCACCCTCTCGGTGGA TTTGAGGAAGCCAACCCGCCAAAGCTTCCCTTGTGTTCAAATCCGCACATGCATTTGTTTGAAGCAATGC TTGCCTGGGAGACTGTCGATCCAGAAACCCAGTTCTGGTCCACATATGCCGATGAGATCGCAAATCTTGC CTTAACGAAATTCATCGATGGCAAGACCGGTGCGTTGCGTGAATTCTTCGATCACGATTGGAAACCTTAT CCGGGTGACAAGGGGAGGGTGGTCGAACCAGGACATCAATTCGAATGGTCCTGGCTGATGGGCCAATGGG CGGACCGACGGCAGAATGGCGATGGCATGATGGCCGCCAAGCGTCTATTCCAGATTGCAGTGGATCGTGG TGTATGCGAGCAGCGCAAAGTTGCCATCATGAGCTTGTATGATGATTTTTCGGTTCACGACAGTCTGGCC CGTCTCTGGCCGCAAACTGAATGGATAAAGTCGGCTCTGTTATTTGCTTCTTTGAGCAAGGACGAAGAGC GAGTTTACTATCTGCAATCCGCATTGCGTGCCATTGACGCCCTACGGCCATTTCTTGAAACGCCAATCAA AGGCTTATGGTACGACAAGTGGCCGGAAGGTGGTACGCTTATCGATGAGCCCGCGCCGGCATCGACCTTT TATCATATTCTCTGTGCCTGTTATGAAGCAGAGAAAACAATATCAGAGCTTTATGTTGTGGTGTCGAATT TTTGGTTGTCGCCGCTGATGATGACGATCAGATGTGCCGATGATGCCGCATCTGTATTGGAGAAACATGC GAGTTCTTCATTTTTTTA >gi|260563601|ref|ZP_05834087.1| ManA family protein [Brucella melitensis bv. 1 str. 16M] MNDVIRKLQVEVERLSSWLHNDALPLWLSAGVDAPNSGFFERIGQDGVATDADNRRSRVHPRQIYCFAKA GAMGWQGEWKQTVEAGIEYYDRVYRREDGFYGSLAGQSGKMIDHTFDLYNQAFSVFAAAQIAVSIPDRFD EMRQRALGLMNTLVADYHHPLGGFEEANPPKLPLCSNPHMHLFEAMLAWETVDPETQFWSTYADEIANLA LTKFIDGKTGALREFFDHDWKPYPGDKGRVVEPGHQFEWSWLMGQWADRRQNGDGMMAAKRLFQIAVDRG VCEQRKVAIMSLYDDFSVHDSLARLWPQTEWIKSALLFASLSKDEERVYYLQSALRAIDALRPFLETPIK GLWYDKWPEGGTLIDEPAPASTFYHILCACYEAEKTISEL Virmugen Unmarked rough B. abortus and B. melitensis deletion mutants of manBA (including manA and manB) were attenuated. These mutants provided protection against virulent Brucella challenge in mice [Ref700:Kahl-McDonagh and Ficht, 2006]. manB Brucella melitensis bv. 1 str. 16M 1198671 17989244 BMEII0899 AE008918 NP_541877 224914 II 940999 942432 + phosphomannomutase 6.8 47237.87 477 5.4.2.8 >gi|17988344:940999-942432 Brucella melitensis bv. 1 str. 16M chromosome II, complete sequence CATGAGCAGCAATTCCCTCAAATTTGGCACGAGCGGCCTTCGCGGGCTGGCGGTAGAACTGAACGGCCTG CCCGCCTATGCCTATACGATGGCCTTTGTGCAGATGCTTGCTGCAAAAGGGCAGTTGCAGAAGGGCGACA AGGTGTTTGTCGGCAGGGATTTGCGCCCCTCCAGCCCTGATATTGCAGCCCTTGCCATGGGTGCCATCGA AGATGCCGGCTTCACACCGGTCAATTGCGGCGTCCTGCCCACGCCTGCGCTGAGCTATTATGCGATGGGC GCGAAAGCACCCAGCATCATGGTCACGGGAAGCCATATTCCAGATGATCGCAACGGGCTGAAATTCTATC GCCGCGACGGTGAAATCGACAAGGATGATGAGGCGGCAATCAGTGCAGCCTATCGCAAGCTGCCTGCCAT TCTCGCTGCCCGCAAACATGTCGGCTCCACCGAAACCGATGCGGCCTTGCAGGCTTATGCCGATCGCTAT GCAGGTTTTCTTGGGAAAGGGAGCCTGAATGGCCTGCGGGTCGGCGTTTATCAACATTCTTCCGTGGCGC GCGATCTTCTGATGTACCTGCTCACGACACTCGGCGTGGAACCCGTGGCGCTCGGACGATCCGATATATT CGTGCCGGTCGATACCGAGGCATTGCGCCCCGAAGACATTGCGCTGCTTGCCCAATGGGGCAAAAGCGAC AGGCTTGATGCCATCGTCTCCACCGACGGAGACGCGGATCGCCCGCTGATTGCCGATGAGCATGGACAAT TCGTTCGCGGCGATCTTGCTGGCGCCATCACCGCCACATGGGTGGGGGCGGATACGCTCGTCACGCCAGT CACCTCCAACACCGCATTGGAAAGCCGCTTTCCCAAGGTTTTGAGAACGCGCGTCGGTTCGCCTTATGTC ATCGCAAGCATGGCACAGGTATCCACGGGCAATTCCGGCCCGGTCATCGGGTTTGAGGCCAATGGCGGCG TTCTGCTTGGCAGCACGGTCGAGAGGAATGGACGAAGCCTGACGGCCCTGCCGACGCGCGACGCCTTGTT GCCCATTCTGGCTTGCCTTGCCACGGTTCACGAAAAGAAAACGCCGCTTTCAACAATCGCCCGGTCCTAT GGCTTCCGCGTCGCGCTTAGCGACCGGCTGCAAAACATTCCGCAGGAGGCGAGCACCGCCTTCCTCGCGC TCTTGGAGGATGCGGATAAACGCGCCTCGCTCTTTCCTGCTGGCGACGCAATCGTGCGGGTGGAAACCAT CGACGGCGTGAAGCTTTTCTTTCAATCAGGCAATGCGGTTCATTATCGGGCATCGGGCAATGCGCCGGAA CTGCGCTGCTATGTGGAATCTTCGGATGACACACAAGCCGCCAAGCTTCAGGCGCTTGGCTTGGAAATCG CACGCAAAGCACTAAAGGATGCGACGAGGCCATG >gi|17989244|ref|NP_541877.1| phosphomannomutase [Brucella melitensis bv. 1 str. 16M] MSSNSLKFGTSGLRGLAVELNGLPAYAYTMAFVQMLAAKGQLQKGDKVFVGRDLRPSSPDIAALAMGAIE DAGFTPVNCGVLPTPALSYYAMGAKAPSIMVTGSHIPDDRNGLKFYRRDGEIDKDDEAAISAAYRKLPAI LAARKHVGSTETDAALQAYADRYAGFLGKGSLNGLRVGVYQHSSVARDLLMYLLTTLGVEPVALGRSDIF VPVDTEALRPEDIALLAQWGKSDRLDAIVSTDGDADRPLIADEHGQFVRGDLAGAITATWVGADTLVTPV TSNTALESRFPKVLRTRVGSPYVIASMAQVSTGNSGPVIGFEANGGVLLGSTVERNGRSLTALPTRDALL PILACLATVHEKKTPLSTIARSYGFRVALSDRLQNIPQEASTAFLALLEDADKRASLFPAGDAIVRVETI DGVKLFFQSGNAVHYRASGNAPELRCYVESSDDTQAAKLQALGLEIARKALKDATRP Virmugen Mutants manB and manA were attenuated in Brucella melitensis and provided protection against challenge in mice's livers [Ref694:Kahl-McDonagh et al., 2007]. mucR Brucella melitensis bv. 1 str. 16M 1197075 17987647 BMEI1364 AE008917 NP_540281 224914 I 1417768 1418253 - transcriptional regulatory protein MUCR 9.56 16771.01 161 >gi|17986284:1417768-1418253 Brucella melitensis bv. 1 str. 16M chromosome chromosome I, complete sequence ATCAGGCGTCCTTCGGCTTGCGGCCGAGGCCCATCTTCTTGGCAAGGCGCGAGCGCGCTGCAGCATAGTT CGGAGCAACCATAGGATAGTTCGGATCGAGATCCCACTTTTCGCGGTACTGCTCAGGCGTCATGTTGTAA TGGGTCACCAGGTGACGCTTCAGCGACTTGAACTTCTTGCCGTCTTCAAGGCAGACAATATAGTCGTCAT GAACAGACTTCTTCGGGTTCACCGCTGGCTTCGGCTTTTCAACAACGACCGGGGCCTCTTCGCGTTCGAC GTGGCGCTTGAAAGCAGCATGAACTTCAGCAATCAGAACCGGGAGTTCGCCTGCACGAATGGAATTATTG CCGACATAGGCTGCAACGACATCGGCGGTCAAACTCAAAAGCAGCTCGGTGCTTTCGTCGTTCGTTTCCA GATTTTCCATAAGTTTTCCTTTTTTAATTTTTTGTTGTCCGCGCAGCGGCTGACAATGGCAACCCA >gi|17987647|ref|NP_540281.1| transcriptional regulatory protein MUCR [Brucella melitensis bv. 1 str. 16M] MGCHCQPLRGQQKIKKGKLMENLETNDESTELLLSLTADVVAAYVGNNSIRAGELPVLIAEVHAAFKRHV EREEAPVVVEKPKPAVNPKKSVHDDYIVCLEDGKKFKSLKRHLVTHYNMTPEQYREKWDLDPNYPMVAPN YAAARSRLAKKMGLGRKPKDA Virmugen A mucR mutant has a significantly reduced degree of colonization in mice and protects against wild type B. melitensis 16M [Ref2071:Arenas-Gamboa et al., 2011]. omp16 Brucella abortus RB51-AHVLA 479804733 CDD:225439 CDD:143586 1198700 ? outer membrane lipoprotein omp16 10.24 18331.05 247 vaccine strain >ENT73703.1 outer membrane lipoprotein omp16 [Brucella abortus RB51-AHVLA] MRRIQSIARSPIAIALFMSLAVAGCASKKNLPNNAGDLGLGAGAATPGSSQDFTVNVGDRIFFDLDSSLI RADAQQTLSKQAQWLQRYPQYSITIEGHADERGTREYNLALGQRRAAATRDFLASRGVPTNRMRTISYGN ERPVAVCDADTCWSQNRRAVTVLNGAGR Protective antigen Omp16 Brucella abortus 2308 VO_0010973 3788229 489056820 BAB_RS24085 AM040264 WP_002966947 359391 I 1654432 1654938 - hypothetical protein 10.24 17438.15 168 Synonyms: Omp16, MotY >NC_007618.1:1654432-1654938 Brucella melitensis biovar Abortus 2308 chromosome I, complete sequence, strain 2308 TTTACCGTCCGGCCCCGTTGAGAACGGTGACGGCGCGACGGTTCTGCGACCAGCATGTGTCGGCATCGCA GACGGCAACCGGGCGCTCATTACCGTAGGAAATGGTGCGCATGCGGTTGGTCGGCACACCGCGCGAAGCG AGGAAGTCGCGGGTGGCGGCAGCACGGCGCTGGCCAAGGGCGAGGTTGTACTCACGCGTGCCGCGCTCGT CGGCATGGCCTTCGATCGTGATCGAATACTGCGGATAACGCTGCAACCACTGGGCCTGCTTGGAAAGCGT CTGCTGCGCATCGGCGCGGATCAGCGACGAATCGAGATCGAAGAAGATGCGGTCGCCGACATTAACGGTG AAGTCCTGCGAGGAGCCCGGCGTTGCAGCGCCTGCACCGAGACCCAGATCACCGGCATTATTCGGAAGGT TCTTCTTTGACGCACAGCCGGCAACGGCGAGCGACATGAAAAGCGCAATAGCGATCGGGCTACGTGCAAT CGACTGGATACGGCGCA >WP_002966947.1 MULTISPECIES: hypothetical protein [Brucella] MRRIQSIARSPIAIALFMSLAVAGCASKKNLPNNAGDLGLGAGAATPGSSQDFTVNVGDRIFFDLDSSLI RADAQQTLSKQAQWLQRYPQYSITIEGHADERGTREYNLALGQRRAAATRDFLASRGVPTNRMRTISYGN ERPVAVCDADTCWSQNRRAVTVLNGAGR Protective antigen Brucella Omp16 is a protective antigen [Ref864:Pasquevich et al., 2009]. Omp19 Brucella abortus 2308 VO_0010972 3788831 489054833 BAB_RS25135 AM040264 WP_002964998 359391 I 1877306 1877839 - hypothetical protein 8.86 16412.87 177 >NC_007618.1:1877306-1877839 Brucella melitensis biovar Abortus 2308 chromosome I, complete sequence, strain 2308 ATCAGCGCGACAGCGTCACGGCCTGCCCGCCGGTGGTCTGGCCATCGAAGCGGCCCTGTCCTGAAGAATA GAGCGAGGCAACCGTACCGCCGTTCGCATCGTAAAGGACGAGTTGCTTGCCATTGACGGCCCAGGAGGCA AGATTAGCCAGTTCACCGGGGCAGCGCAGCGGGCCTGCGCGATAGCCCTGGCCATATTTGGTCTGCGGCG TCGCGATCTTGCAGCTCTGACCACCAAGCGAGGCGTTCCAGACGCCAGCCACGGCGCCGGGCGTCAGGTC CGGTGCGGATGCAGGCGGCAGGCTTGCGACCTGTGTGCCGGATTGCGCGCTCATATCCGTGGAGGGCGCA TTGGGGAATTGTGTGGGAGAATCAAGATTGCCTTTCTGCACCGTGCCTGCCGGAACAGCATTGACCGGTG CAGGCGGCGGCGGAGGCGAAACATTATCGAGATTACCAAGCCGGGAGCTCTGGCACCCGGCCAGGACAAT GCCAGCCGCCGCGAGGCTGAGCAGACTTGCTTTTGAAATTCCCA >WP_002964998.1 MULTISPECIES: hypothetical protein [Brucella] MGISKASLLSLAAAGIVLAGCQSSRLGNLDNVSPPPPPAPVNAVPAGTVQKGNLDSPTQFPNAPSTDMSA QSGTQVASLPPASAPDLTPGAVAGVWNASLGGQSCKIATPQTKYGQGYRAGPLRCPGELANLASWAVNGK QLVLYDANGGTVASLYSSGQGRFDGQTTGGQAVTLSR Protective antigen Brucella Omp19 is a protective antigen [Ref864:Pasquevich et al., 2009]. Omp25 Brucella melitensis bv. 1 str. 16M VO_0010966 1196960 17987532 BMEI1249 AE008917 NP_540166 224914 I 1297524 1298165 - hypothetical protein 9.03 21696.72 213 Reference: http://www.ncbi.nlm.nih.gov/pubmed/17049676 >NC_003317.1:1297524-1298165 Brucella melitensis bv. 1 str. 16M chromosome I, complete sequence ATTAGAACTTGTAGCCGATGCCGACGCGGAAATCCTGCGTGTCCAGCTTGTTGCGAACAGTCGTACCGGC CAGATCATAGTTCTTGTTGCCGTACTGGGTGTAACGGTACTCAACGCGGCCGAGGATGTTGTCCGTCAGC TTGGCTTCGAGACCGGCACCAGCCGTCCAACCCACGCGGAACTTGCTTTCGTCGTCCAAGCCGTTGTTAA GCTTGATCTGCGAACCGGCAATACCAGCCGTGAGGTACGGCATAACCGGGTTCAGGTCGTAGCCAACGCG GGCACGCAGCGAGCCTTCAAAGCCCTGCTTGACTTCCAGGCCGTCCTTGGACTTCTTGGCCCAGGAATAA CCTGCATCACCTTCAACACCGTATACGATCTGGTCCTGCTGGAAGTTCCAGCCAGCAAAGGCGCCAGCCT TCCAATCGTCAGGCTTGATGCTGCCAACGGTGCTGGTCTTGGCCTTGTTCCAGCCGTAGCCAAGGTAAAG ACCGGTATAGCCACCAGCCCAGCTATACTGGGGAGCTACTTCAACCGGAGCCGGAACCGGAGGCTGTTCC TGGATGGCGTCGGCAGCAAAAGCGGTCGCAGAGAACGGCAGCAACGCAGCCGAGACGATTACGAGAGACT TAAGAGTGCGCA >NP_540166.1 hypothetical protein BMEI1249 [Brucella melitensis bv. 1 str. 16M] MRTLKSLVIVSAALLPFSATAFAADAIQEQPPVPAPVEVAPQYSWAGGYTGLYLGYGWNKAKTSTVGSIK PDDWKAGAFAGWNFQQDQIVYGVEGDAGYSWAKKSKDGLEVKQGFEGSLRARVGYDLNPVMPYLTAGIAG SQIKLNNGLDDESKFRVGWTAGAGLEAKLTDNILGRVEYRYTQYGNKNYDLAGTTVRNKLDTQDFRVGIG YKF Protective antigen Brucella Omp25 is a protective antigen [Ref863:Commander et al., 2007]. Virmugen A omp25 mutant is attenuated and provides protection against challenge in mice [Ref1946:Edmonds et al., 2002]. omp31 Brucella melitensis ATCC 23457 7676420 225853091 BMEA_A1674 CP001488 YP_002733324 546272 I 1588634 1589242 + outer membrane protein Omp31 4.8 20497.92 202 >gi|225851546:1588634-1589242 Brucella melitensis ATCC 23457 chromosome chromosome I, complete sequence TGTGTTGCAATATAGTGACAGACATTGGAGCCCATATTGGGCCCAAGATTTTTCAGGTTCGCTCGACGTC ACGGCCAGCGGCTTTGTTGGCGGCGTTCAGGCCGGTTATAACTGGCAGCTTGCCAACGGCCTCGTGCTTG GTGGCGAAGCTGACTTCCAGGGCTCGACGGTTAAGAGCAAGCTTGTTGACAACGGTGACCTCTCCGATAT CGGCGTTGCAGGCAACCTCAGCGGCGACGAAAGCTTCGGCCTCGAGACCAAGGTTCAGTGGTTTGGAACG GTGCGTGCGCGCCTCGGCTTCACCCCGACTGAACGCCTGATGGTCTATGGTACCGGTGGTTTGGCCTATG GTAAGGTCAAGACGTCGCTTAGCGCCTATGACGATGGTGAATCGTTCAGCGCCGGAAACTCTAAGACCAA GGCTGGCTGGACGCTTGGTGCAGGTGTAGAATACGCCGTCACCAACAATTGGACCCTGAAGTCGGAATAC CTCTACACCGACCTCGGCAAGCGTTCCTTCAATTACATTGATGAAGAAAACGTCAATATTAACATGGAAA ACAAGGTGAACTTCCACACCGTCCGCCTCGGTCTGAACTACAAGTTCTA >gi|225853091|ref|YP_002733324.1| outer membrane protein Omp31 [Brucella melitensis ATCC 23457] MLQYSDRHWSPYWAQDFSGSLDVTASGFVGGVQAGYNWQLANGLVLGGEADFQGSTVKSKLVDNGDLSDI GVAGNLSGDESFGLETKVQWFGTVRARLGFTPTERLMVYGTGGLAYGKVKTSLSAYDDGESFSAGNSKTK AGWTLGAGVEYAVTNNWTLKSEYLYTDLGKRSFNYIDEENVNINMENKVNFHTVRLGLNYKF Virmugen A omp31 mutant in Brucella melitensis was attenuated and provided protection against challenge in mice [Ref1988:Cloeckaert et al., 2004]. omp31 Brucella melitensis bv. 1 str. 16M 61229373 CDD:226163 224914 ? 31 kDa outer-membrane immunogenic protein 5.06 24732.46 343 biovar: 1 str. 16M >sp|P0A3U4.1|OM31_BRUME RecName: Full=31 kDa outer-membrane immunogenic protein; Flags: Precursor MKSVILASIAAMFATSAMAADVVVSEPSAPTAAPVDTFSWTGGYIGINAGYAGGKFKHPFSSFDKEDNEQ VSGSLDVTAGGFVGGVQAGYNWQLDNGVVLGAETDFQGSSVTGSISAGASGLEGKAETKVEWFGTVRARL GYTATERLMVYGTGGLAYGKVKSAFNLGDDASALHTWSDKTKAGWTLGAGAEYAINNNWTLKSEYLYTDL GKRNLVDVDNSFLESKVNFHTVRVGLNYKF Protective antigen Omp31 Brucella abortus 2308 VO_0010939 3788834 489054549 BAB_RS23735 AM040264 WP_002964719 359391 I 1583642 1584427 + membrane protein 4.74 25591.47 261 >NC_007618.1:1583642-1584427 Brucella melitensis biovar Abortus 2308 chromosome I, complete sequence, strain 2308 CATGTTTAGCTTAAAAGGGACTGTTATGAAAACCGCACTTCTTGCATCCGTCGCAATGTTGTTCACAAGC TCGGCTATGGCTGCCGACATCATCGTTGCTGAACCGGCACCCGTTGCAGTCGACACGTTCTCTTGGACTG GCGGCTATATTGGTATCAATGCTGGTTACGCTGGCGGCAAGTTCAAGCATCCGTTCTCAGGCATCGAGCA GGATGGGGCCCAAGATTTTTCAGGTTCGCTCGACGTCACGGCCAGCGGCTTTGTTGGCGGCGTTCAGGCC GGTTATAACTGGCAGCTTGCCAACGGCCTCGTGCTTGGTGGCGAAGCTGACTTCCAGGGCTCGACGGTTA AGAGCAAGCTTGTTGACAACGGTGACCTCTCCGATATCGGCGTTGCAGGCAACCTCAGCGGCGACGAAAG CTTCGTCCTCGAGACCAAGGTTCAGTGGTTTGGAACGGTGCGTGCGCGCCTCGGCTTCACCCCGACTGAA CGCCTGATGGTCTATGGTACCGGTGGTTTGGCCTATGGTAAGGTCAAGACGTCGCTTAGCGCCTATGACG ATGGTGAATCGTTCAGCGCCGGAAACTCTAAGACCAAGGCTGGCTGGACGCTTGGTGCAGGTGTAGAATA CGCCGTCACCAACAATTGGACCCTGAAGTCGGAATACCTCTACACCGACCTCGGCAAGCGTTCCTTCAAT TACATTGATGAAGAAAACGTCAATATTAACATGGAAAACAAGGTGAACTTCCACACCGTCCGCCTCGGTC TGAACTACAAGTTCTA >WP_002964719.1 MULTISPECIES: porin family protein [Brucella] MFSLKGTVMKTALLASVAMLFTSSAMAADIIVAEPAPVAVDTFSWTGGYIGINAGYAGGKFKHPFSGIEQ DGAQDFSGSLDVTASGFVGGVQAGYNWQLANGLVLGGEADFQGSTVKSKLVDNGDLSDIGVAGNLSGDES FVLETKVQWFGTVRARLGFTPTERLMVYGTGGLAYGKVKTSLSAYDDGESFSAGNSKTKAGWTLGAGVEY AVTNNWTLKSEYLYTDLGKRSFNYIDEENVNINMENKVNFHTVRLGLNYKF Protective antigen ORF VO_0010927 599653 599654 CAA86936.1 1DI0 CDD:237096 CDD:187741 GOA:P61711 InterPro:IPR002180 PDB:1DI0 PDB:1T13 PDB:1XN1 UniProtKB/Swiss-Prot:P61711 235 ? 7.41 16119.15 197 riboflavin synthase subunit beta; Provisional >gi|599653|emb|Z46864.1| B.abortus gene for open reading frame GAATTCCGATCAGTGCATAGTTTCCGCGTGCTCGCGCAATGGTGCGCGGGCTTGTTCTCGGGGCGGGGTG AAACTCCCCACCGGCGGTATGAAAAGCAATTTTCAAGCCCGCGAGCGCCTGAAATGGAAGCCGATTCGCA TGCCATTTCAGGGTCAGCAGATCCGGTGAGATGCCGGAGCCGACGGTTAAAGTCCGGATGGAAGAGAGCG AATGAGCGTCACGATTGCGCCTTCCGGCGTCGTTCTTGCGTTCTTTTGTGCGCCCTGATTCTAGTTTCGT GAGGAACCTATGAACCAAAGCTGTCCGAACAAGACATCCTTTAAAATCGCATTCATTCAGGCCCGCTGGC ACGCCGACATCGTTGACGAAGCGCGCAAAAGCTTTGTCGCCGAACTGGCCGCAAAGACGGGTGGCAGCGT CGAGGTAGAGATATTCGACGTGCCGGGTGCATATGAAATTCCCCTTCACGCCAAGACATTGGCCAGAACC GGGCGCTATGCAGCCATCGTCGGTGCGGCCTTCGTGATCGACGGCGGCATCTATCGTCATGATTTCGTGG CGACGGCCGTTATCAACGGCATGATGCAGGTGCAGCTTGAAACGGAAGTGCCGGTGCTGAGCGTCGTGCT GACGCCGCACCATTTCCATGAAAGCAAGGAGCATCACGACTTCTTCCATGCTCATTTCAAGGTGAAGGGC GTGGAAGCGGCCCATGCCGCCTTGCAGATCGTGAGCGAGCGCAGCCGCATCGCCGCGCTTGTCTGACTAA CCCTCTATAATACGCCCGCAATGGGTATAAATGTCGAATTC >CAA86936.1 ORF [Brucella abortus] MNQSCPNKTSFKIAFIQARWHADIVDEARKSFVAELAAKTGGSVEVEIFDVPGAYEIPLHAKTLARTGRY AAIVGAAFVIDGGIYRHDFVATAVINGMMQVQLETEVPVLSVVLTPHHFHESKEHHDFFHAHFKVKGVEA AHAALQIVSERSRIAALV Protective antigen This gene was originally isolated from a lambda gt11 expression library probed with sera from Brucella-infected sheep. Sequence analysis of the cloned gene revealed the presence of an open reading frame of 158 amino acids encoding a protein of 17.3 kDa [Ref417:Hemmen et al., 1995]. It was later identified as a cytoplasmic protein lumazine synthase [Ref418:Goldbaum et al., 1999]. This gene can be used for diagnosis [Ref417:Hemmen et al., 1995] [Ref418:Goldbaum et al., 1999] and vaccine development [Ref416:Velikovsky et al., 2002]. P39 from Brucella abortus Brucella abortus bv. 1 str. 9-941 68067499 CDD:224567 262698 ? Probable sugar-binding periplasmic protein 5.06 44330.68 589 biovar: 1 str. 9-941 >sp|O06875.3|SP39_BRUAB RecName: Full=Probable sugar-binding periplasmic protein; AltName: Full=Immunogenic 39 kDa protein; AltName: Full=P39; Flags: Precursor MHKLLKLAAMGTAACALLAGMAPVANAQEKQNVEVLHWWTSGGEASALEVLKKDLESKGISWTDMPVAGG GGTEAMTVLRARVTAGNAPTAVQMLGFDIRDWAEQGALGNLDTVASKEGWEKVIPAPLQEFAKYDGHWIA APVNIHSTNWMWINKAALDKAGGKEPTNWDELIALLDNFKAQGITPIAHGGQPWQDATIFDAVVLSFGPD FYKKAFIDLDPEALGSDTMKQAFDRMSKLRTYVDDNFSGRDWNLASAMVIEGKAGVQFMGDWAKGEFLKA GKKPGEDFVCMRYPGTQGAVTFNSDMFAMFKVSEDKVPAQLEMASAIESPAFQSAFNVVKGSAPARTDVP DTAFDACGKKAIADVKEANSKGTLLGSMAHGYANPAAVKNAIYDVVTRQFNGQLSSEDAVKELVAAVEAA K Protective antigen pgk Brucella melitensis biovar Abortus 2308 3788256 82700518 BAB1_1742 AM040264 YP_415092 359391 I 1682955 1684142 ? phosphoglycerate kinase 5.91 37696.73 395 Converts 3-phospho-D-glycerate to 3-phospho-D-glyceroyl phosphate during the glycolysis pathway >gi|82698932:1682955-1684142 Brucella melitensis biovar Abortus 2308 chromosome chromosome I, complete sequence GATGTTCCGCACCCTTGACGATGCCAATGTCCAATCCAAGCGCGTGCTGGTCCGTGTTGACCTCAACGTG CCGATGGCAAACGGCGAAGTCACCGACCTTACCCGCATCGAACGCATCGTTCCGACCATCGCGGAACTGT CAAGGAAGGGCGCGAAGGTCATCCTGCTCGCCCATTTCGGTCGCCCGAAGGGGGTTGCCTCGGATGAAAA TTCGCTGAAGCATGTCGTGAAGCCGCTCTCCAAGGTTCTGGATCACAGTGTTCATTTTGCCGAAGACTGC ATCGGCGACAAGGCGAAAGCGGCCGTCGATGCGCTGAAGGACGGCGACGTCCTGCTTCTGGAAAATACCC GTTTCCACAAGGGCGAGGAAAAGAACGATCCTGAATTCGTGCAGGCGCTGGCCGCCAATGGCGATCTCTA TGTGAACGATGCCTTTTCCGCCGCTCACCGCGCCCATGCCTCGACTGAAGGTCTGGCCCATGTCCTGCCT GCCTTCGCGGGCCGCGCCATGCAGGCTGAGCTTGAAGCACTTGAAAAGGGCCTCGGCAATCCGGCCCGCC CGGTGGTCGCCATCGTCGGCGGTGCAAAGGTTTCGACCAAGCTCGACCTCCTGTCCAACCTGATCGAAAA GGTCGATGCGCTTGTCATCGGCGGCGGCATGGCCAACACGTTCCTTGCAGCCAAGGGTCTTGATGTCGGC AAATCGCTCTGCGAGCATGAGCTGGCCTCGACCGCACGCGAAATCATGGCCAAGGCCGAAACCACCAAAT GCGCCATCATCCTGCCGGTGGACGCGGTCGTGGGCTGGCACTTCGCCGCCGATACGCCACACCAGACCTA TGGCGTGGATTCGGTTCCGGGCGACGCCATGATCCTTGACGCAGGCGAGCTTTCGACCGACCTCATTGCT TCGGCCATCGATGATGCCGCAACATTGGTCTGGAACGGCCCGCTTGGGGCTTTCGAGTTGCGCCCCTTTG ACACAGCAACGGTGAAGGTCGCAAGGCATGTCGCAAAGCGCACAAAGGAAGGCAAGCTTGTTTCGGTCGG CGGCGGCGGCGATACGGTGGCAGCCCTCAACCATGCGGGCGTGGCCGACGACTTCACCTATATCTCAACC GCTGGCGGCGCTTTCCTCGAATGGATGGAAGGCAAACCGCTTCCGGGCGTCGATGTATTGAAGAAGTG >gi|82700518|ref|YP_415092.1| phosphoglycerate kinase [Brucella melitensis biovar Abortus 2308] MFRTLDDANVQSKRVLVRVDLNVPMANGEVTDLTRIERIVPTIAELSRKGAKVILLAHFGRPKGVASDEN SLKHVVKPLSKVLDHSVHFAEDCIGDKAKAAVDALKDGDVLLLENTRFHKGEEKNDPEFVQALAANGDLY VNDAFSAAHRAHASTEGLAHVLPAFAGRAMQAELEALEKGLGNPARPVVAIVGGAKVSTKLDLLSNLIEK VDALVIGGGMANTFLAAKGLDVGKSLCEHELASTAREIMAKAETTKCAIILPVDAVVGWHFAADTPHQTY GVDSVPGDAMILDAGELSTDLIASAIDDAATLVWNGPLGAFELRPFDTATVKVARHVAKRTKEGKLVSVG GGGDTVAALNHAGVADDFTYISTAGGAFLEWMEGKPLPGVDVLKK Virmugen A pgk mutant is attenuated in mice and induces significant protection from challenge with wild type B. abortus [Ref1811:Trant et al., 2010]. pgm Brucella melitensis biovar Abortus 2308 3788847 82698981 BAB1_0055 AM040264 YP_413555 359391 I 60852 62483 - phosphoglucomutase 6.52 55523.82 543 catalyzes the interconversion of alpha-D-glucose 1-phosphate to alpha-D-glucose 6-phosphate >gi|82698932:60852-62483 Brucella melitensis biovar Abortus 2308 chromosome chromosome I, complete sequence CTCAAGTAACCACCGACGGCTCTGTCCGCCCGCTGCGTTTTTTTACTTCGGCGATCTGTTCAGCCGCGTC GATCAGTGGGGCCAGCGTTGCCTGCGTGTCGAGATTATGTTTTGCAGGATCGGCTTCATAGCGTTCGATA TAGATGCGGATGGTCGCGCCTGACGTCCCGGTACCGGACAGGCGAAGCACGATGCGCGCACCTCCTTCAA AATAGATGCGGATGCCCTGATGTTCGCTGGTCGAGCCATCAACGGGATCATGATAGGCGAAGTCGTCGGC CTTTTCGATCCTGAGGCCATTGACGCTCGTGCCGGGCAGGCCGGCAAGCTTGCCGCGCAGGTCAGCCACC AGCTTCGTCGCAATATCGGAATCGACGGCTTCATAGTCGTGGCGAGTGTAATAATTGCGCCCGAAACGTG CCCAATGGTCGTCCGCGATTGCCTTCACACTTTCCTTGCGCACCGCCAGAATGTTGAGCCAGAGCAGAAC GGCCCACAAACCATCTTTTTCGCGCACATGGTCGGAGCCGGTGCCGGAGCTTTCCTCGCCGCAGATCGTC ACCTTGCCGCTGTCGAGAAGATTGCCGAAGAATTTCCAGCCGGTCGGGGTTTCATACATGCCGATACCGA GTTTTTCCGCCACGCGGTCGGCGGCGGCGCTCGTCGGCATGGAACGGGCAATGCCCTTGATACCGCCTTT ATAGCCCGGCGCCAGATGGGCATTTGCGGCCAGCATGGCAAGCGAATCTGACGGCGTGATGAAGATACCG CGCCCGAGAATGAGGTTGCGATCGCCATCGCCGTCGGAGGCGGCGCCAAAATCCGGCGCATGGCTGGACA TCAGAAGATCGTAGAGATCCTTGGCATAGACGAGGTTTGGATCCGGGTGATGGCCACCGAAATCCGGCAG GGGCACGAAATTGACGACGCTGCCTTCGGGCGCACCGAGGCGGCGCTCGAAGATTTCCTTGGCATAAGGC CCGGTCACCGCATGCATGGCGTCGAACTTCATCTGGAAGCCGCCTTTTATCATGGCGCGGATGGCGGCGA AATCGAACAGGCTTTCCATCAATTCAGCATAATCGGCCACCGGATCGAAGATGACCACCTCGGTATCGCC GATCTTGCTTGTGCCGATCTTGTCGATATCGATATCGGCTGCATCTGCAATTTTATACTGGTCGATCACC TTTGAGCGGGCGAAGATCGCTTCGGTGATCTTTTCGGGTGCCGGTCCACCATTGCCGATATTGTACTTGA TGCCGAAATCCTGCGTAGGCCCGCCCGGATTGTGGCTGGCCGAAAGGATCATGCCACCGAAGGCTTTGTA CTTGCGGATCATGTTGGAGGCGGCTGGCGTGGAAAGAATGCCGCCCTGTCCCACCATGATACGGCCAAAA CCGTTGGCGGCTGCGATCTTGATAAGCTTCTGGATCACCTCGCGGTTATAAAAGCGTCCGTCGCCGCCAA CCACGAGCGTCTTGCCCTTGAACCCCTCCAGCACGTCGAAGATCGACTGGATGAAATTTTCTGCATAATT GGGCTGCTGGAAAACCGGAACCTTCTTGCGCAAGCCCGAAGTGCCCGGCTGTTGATCCTGGTATGGCGTG GTCGCAATGGTCTTGACGGTCA >gi|82698981|ref|YP_413555.1| phosphoglucomutase [Brucella melitensis biovar Abortus 2308] MTVKTIATTPYQDQQPGTSGLRKKVPVFQQPNYAENFIQSIFDVLEGFKGKTLVVGGDGRFYNREVIQKL IKIAAANGFGRIMVGQGGILSTPAASNMIRKYKAFGGMILSASHNPGGPTQDFGIKYNIGNGGPAPEKIT EAIFARSKVIDQYKIADAADIDIDKIGTSKIGDTEVVIFDPVADYAELMESLFDFAAIRAMIKGGFQMKF DAMHAVTGPYAKEIFERRLGAPEGSVVNFVPLPDFGGHHPDPNLVYAKDLYDLLMSSHAPDFGAASDGDG DRNLILGRGIFITPSDSLAMLAANAHLAPGYKGGIKGIARSMPTSAAADRVAEKLGIGMYETPTGWKFFG NLLDSGKVTICGEESSGTGSDHVREKDGLWAVLLWLNILAVRKESVKAIADDHWARFGRNYYTRHDYEAV DSDIATKLVADLRGKLAGLPGTSVNGLRIEKADDFAYHDPVDGSTSEHQGIRIYFEGGARIVLRLSGTGT SGATIRIYIERYEADPAKHNLDTQATLAPLIDAAEQIAEVKKRSGRTEPSVVT Virmugen A pgm mutant is attenuated in mice and induces significant protection from challenge with wild type B. abortus [Ref1909:Ugalde et al., 2003]. purE Brucella melitensis bv. 1 str. 16M 600729 BMEI0295 NZ_GG703781 NP_539212 CDD:200634 29459 I 308233 309321 - 5'-phosphoribosyl-5-amino-4-imidazole carboxylase 5.29 36300.44 161 AIR carboxylase; cl00310 >gi|17986284:308233-309321 Brucella melitensis bv. 1 str. 16M chromosome chromosome I, complete sequence CTTAAATTGTGCGGGGCTTTATGCGGGTCACATGGCCTATTTTCCGGCCCGCGCGAGCTTCCTTTTTACC GTAAAGATGCAGCACGGCGTTCTTCTCGCAGAGAATCGCCGGAACCTTTTCGATATCGTCGCCAATCAGG TTTTCCATCACACAGTCGCTATGGCGATCCGTATTGCCGAGCGGCAGTCCCGCCACAGCGCGGATATGCT GCTCAAATTGGGAAATGGCGCAGGCTGCTTCCGTCCAGTGGCCCGAATTATGCACACGCGGGGCAAATTC ATTGGCGAGCAGCGTGCCGTCCTTCAGCACGAAGAATTCAAGCCCCAGCACACCGACATAGTCCAGCGCG TGCAACAGTTTTTCGGCGGCTGTGCGCGCGGCTTCTGCCGTCTGTACGCTGATCGCGGCAGGCACTGTGG ACGTGGCGAGAATGCCATCCTTGTGGACGTTTTCCGCAAGATCGAAGATGGCGACATTGCCGCTGCGATC GCGCGCGGCGATGACGGAGACTTCGCGCTCGAATTCCACGAAGCCTTCGAGAATCGCAGGCGCCTTGTTG ATGGCTGCAAAAGCGTTGCAGGCCTGGGTTTCATCGAGGGAGGCAAGGCGCACCTGCCCCTTGCCGTCAT AACCCAGACGCCGTATCTTGAGGATGCCACGCCCGCCCAGTGCGCCGAGCGCGGCGATGAGCGTTTCCTC GTCATCCACGAGCCGCCAGGGCGCGGTTTCAATGCCGCTTTCGTTGAGAAACTGCTTTTCTGTGAAGCGG TCCTGAGAGATTTCCAGTGCGGCGGGCGGGGGCAGAACAAGCGCCGTTTCAGCCAGCTTGTCGGCGGCGC TGACTGGCACATTTTCAAATTCATAGGTGATGACGTCGGAAGCGGCGGCAAGTTCGGCCAGCGCCTTCGG GTCATCATAGGCGGCGACAATCTGGCGATTGGCAACCTGTGCTGCCGGGCAACCGGCCTGCGGCTCAAGG ATTATGGTTTCATAACCGAAGCGCGCCGCTGCCATGGCGAGCATACGGCCAAGCTGGCCCCCGCCGATAA TGCCGATGGTGGAGCCGGGCTTGAGAGATGTCTTGTCCA >gi|600729|gb|AAA57002.1| 5'-phosphoribosyl-5-amino-4-imidazole carboxylase [Brucella melitensis] MSVDVAIIMGSQSDWETMHHAADTLEALGISFDARIVSAHRTPDRLVAFAKGAKREGFKVIIAGAGRAHL PGMAAAMTPLPVFGVPVQSKALSGQDSLLSIVQMPAGIPVGTLAIGRAGAVNAALLAAAVLALYDEALAA RLDEWRKAQTESVAERPSNEA Virmugen Mice were infected with delta purE201 or wild type parent strain 16M. The number of bacteria in the spleen and spleen weight peaked for both strains between 1 and 2 weeks postinfection (p.i.), though the number of delta purE201 cells was significantly less than the number of 16M cells recovered from the spleens of infected mice [Ref1799:Crawford et al., 1996]. Later, a purE/purK mutant demonstrated protection from challenge with wild type B. melitensis [Ref704:Hoover et al., 1999]. purK Brucella melitensis bv. 1 str. 16M 20141763 CDD:180352 CDD:200773 CDD:194530 224914 I ? N5-carboxyaminoimidazole ribonucleotide synthase 362 biovar: 1 str. 16M >gi|3788877|gb|AQ262397.1|AQ262397 CITBI-E1-2502P3.TF CITBI-E1 Homo sapiens genomic clone 2502P3, DNA sequence CACCACCCCCCAGCCTGAGACCAAAGTCACAGTTTAACTCTGTCATGGGTCATGTGCAGAGGGAAGGGAC TGCCCCAGAATTAGCAGTTGGCTGAACACCCTGCTTAAAAACTTTTTCTTTTCCTTTTTTTGTGAGTACC GTCCGCTTGGACTAAAGAAGTTTTGGTTCGTTGTTTTCTTAATTTTGGGCTACAGAGTCTCTACCTACAT TTGTGTTTTGTGTTTTGGTAACCACATGTAACAGCCGGTGGAATCTTTTTTCTCTTTGCTTTCTCTGAAC TTTTCAAAGAGTAGGCCCCTATCTTTCGGGGGATTAACTCCTTGTGGCCCCTGTGTCGTTGGTGCT >gi|20141763|sp|P52559.2|PURK_BRUME RecName: Full=N5-carboxyaminoimidazole ribonucleotide synthase; Short=N5-CAIR synthase; AltName: Full=5-(carboxyamino)imidazole ribonucleotide synthetase MDKTSLKPGSTIGIIGGGQLGRMLAMAAARFGYETIILEPQAGCPAAQVANRQIVAAYDDPKALAELAAA SDVITYEFENVPVSAADKLAETALVLPPPAALEISQDRFTEKQFLNESGIETAPWRLVDDEETLIAALGA LGGRGILKIRRLGYDGKGQVRLASLDETQACNAFAAINKAPAILEGFVEFEREVSVIAARDRSGNVAIFD LAENVHKDGILATSTVPAAISVQTAEAARTAAEKLLHALDYVGVLGLEFFVLKDGTLLANEFAPRVHNSG HWTEAACAISQFEQHIRAVAGLPLGNTDRHSDCVMENLIGDDIEKVPAILCEKNAVLHLYGKKEARAGRK IGHVTRIKPRTI Virmugen A purE/purK mutant of B. melitensis 16M was attenuated in mice when given intraperitoneally. Nine weeks after immunization, mice were protected from challenge with wild type B. melitensis [Ref706:Drazek et al., 1995]. RL3382 Rhizobium leguminosarum bv. viciae 3841 4399754 116253124 RL3382 NC_008380 YP_768962 216596 3553339 3554079 ? putative outer membrane protein related to bp26 antigen of Brucella 4.91 23778 246 similarity:fasta; with=UniProt:Q5W9U9_9RHIZ (EMBL:AB126349); Brucella sp. JM13/00.; bp26; Outer membrane protein Bp26.; length=250; id 51.515; 231 aa overlap; query 20-245; subject 21-250~similarity:fasta; with=UniProt:Q92NA6_RHIME (EMBL:SME591790); Rhizobium meliloti (Sinorhizobium meliloti).; Hypothetical protein SMc01556.; length=262; id 58.594; 256 aa overlap; query 7-246; subject 8-262 >gi|4399754|gb|AI501903.1|AI501903 UI-R-C2p-sd-g-11-0-UI.s1 UI-R-C2p Rattus norvegicus cDNA clone UI-R-C2p-sd-g-11-0-UI 3', mRNA sequence TTTTTTTTTTTTTTATTCGGTTTTATTTATTTATTTTTGGTGCAGACAAGAAACATAACAGTAACATGTC TTTCCTTCGAGCACAAAGAAGACAGAGGACTAAGATCCTCATGTAGTTGCTAAGCAGTTCTAGTCCTCAC ACTGGTTAAGAACAAAATGTCAAGTATTACTACAGCAAGAAATATCAGTAAGACAAGTTACATTTGTCTT TTTGAAACCACTTTTATAAGACATAGCCACAGTTCTCAAAAGTTCTTCAGACTTCAGAGATCACATTGTA CGGATTGTACTTCTAAGGATACAGCTATACGTGAACAGACACCAGAACAAGTTAATCTAGTAATGAATAT TCACAGGAGTCTTCATACTGCAAATGACAGAAATTCCAACGTTTGCACTGTTCTCAGCG >gi|116253124|ref|YP_768962.1| putative outer membrane protein related to bp26 antigen of Brucella [Rhizobium leguminosarum bv. viciae 3841] MAPIYTKTVLMTALLALSLAAAAPAFAQEAKPREPVISVTGDGESSVAPDMAIVNLAVVKQAKTAREALD ENNKAMNDVLAALKSGGIAERDLQTSGFSIQPQYNYPQPVDGQQQQPQLIGYQTINSVTVRLRDLAKLGA VIDQSVTLGINQGGEIQFTNDKPDAAIEAARKAAVADAVKRAKTLSEAAGVKLGRILEINENVPRAMPQP VYRATMMKEASDAAVPVQGGENNYNVSVTVTFAIEQ RplL Brucella abortus 2308 VO_0010908 3788918 489054195 BAB_RS21965 AM040264 WP_002964371 359391 I 1229918 1230292 - 50S ribosomal protein L7/L12 4.49 11356.05 124 present in two forms; L12 is normal, while L7 is aminoacylated at the N-terminal serine; the only multicopy ribosomal protein; 4:1 ratio of L7/L12 per ribosome; two L12 dimers bind L10; critically important for translation efficiency and fidelity; stimulates GTPase activity of translation factors >NC_007618.1:1229918-1230292 Brucella melitensis biovar Abortus 2308 chromosome I, complete sequence, strain 2308 CTTACTTGAGTTCAACCTTGGCGCCAGCAGCTTCGAGCTGTGCCTTGATCTTCTCAGCTTCGTCCTTCGA GGCGCCTTCCTTGACAGCCTTCGGAGCGCCTTCGACCAGGTCCTTGGCTTCCTTGAGGCCGAGACCGGTG AGTGCGCGCACTTCCTTGATCACGTTGATCTTGTTAGCGCCGCCGTCAGCGAGAACGACGTCGAATTCGG TCTTTTCTTCTGCGGCAGCAGCAGGGGCAGCGCCACCGGCAGCAGCAACAGCGACCGGAGCAGCAGCCGA AACGCCCCACTTCTCTTCGAGAAGCTTGGACAGCTCAGCGGCTTCCAGAACGGTCAGGGCCGAAAGGTCT TCAACGATCTTTGCGAGATCAGCCA >WP_002964371.1 MULTISPECIES: 50S ribosomal protein L7/L12 [Brucella] MADLAKIVEDLSALTVLEAAELSKLLEEKWGVSAAAPVAVAAAGGAAPAAAAEEKTEFDVVLADGGANKI NVIKEVRALTGLGLKEAKDLVEGAPKAVKEGASKDEAEKIKAQLEAAGAKVELK Protective antigen Study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein, rplL) and Omp16 protein (outer membrane lipoprotein). This divalent DNA vaccine induced a significant level of protection against challenge with the virulent B. abortus in BALB/c mice [Ref697:Luo et al., 2006b]. RpsL Brucella melitensis bv. 1 str. 16M VO_0010963 1196463 161511151 BMEI0752 NZ_GG703780 NP_539669 224914 I 782951 783322 + 30S ribosomal protein S12 12.15 12482.71 123 interacts with and stabilizes bases of the 16S rRNA that are involved in tRNA selection in the A site and with the mRNA backbone; located at the interface of the 30S and 50S subunits, it traverses the body of the 30S subunit contacting proteins on the other side; mutations in the S12 gene confer streptomycin resistance >gi|17986284:782951-783322 Brucella melitensis 16M chromosome I, complete sequence AATGCCTACCGTAAACCAGCTTATCCGCAAGCCGCGCACTGCGCCGGTGAAGCGCAATAAGGTTCCTGCA CTGCAGGCAAACCCTCAGAAGCGCGGCGTTTGCACCCGCGTTTACACGACGACCCCGAAGAAGCCGAACT CGGCTCTGCGTAAGGTTGCCAAGGTTCGTCTGACGAACGGCTTTGAAGTCATTGGTTATATCCCTGGTGA AGGGCATAACCTGCAGGAGCACTCCGTCGTGATGATCCGTGGCGGTCGTGTGAAGGACTTGCCGGGTGTG CGTTACCACATCATCCGTGGCGTTCTCGATACCCAGGGTGTCAAGAACCGCAAGCAGCGCCGTTCCAAGT ACGGTGCAAAGCGTCCGAAGTA >gi|161511151|ref|NP_539669.2| 30S ribosomal protein S12 [Brucella melitensis 16M] MPTVNQLIRKPRTAPVKRNKVPALQANPQKRGVCTRVYTTTPKKPNSALRKVAKVRLTNGFEVIGYIPGE GHNLQEHSVVMIRGGRVKDLPGVRYHIIRGVLDTQGVKNRKQRRSKYGAKRPK Other SodC from B. abortus strain 2308 Brucella abortus 2308 VO_0010856 3827840 489062065 BAB_RS28905 AM040265 WP_002972093 2AQM 359391 II 534068 534589 + superoxide dismutase [Cu-Zn] 6.74 16741.77 173 >NC_007624.1:534068-534589 Brucella melitensis biovar Abortus 2308 chromosome II, complete sequence, strain 2308 GATGAAGTCCTTATTTATTGCATCGACAATGGTGCTTATGGCTTTTCCGGCTTTCGCAGAAAGCACGACG GTAAAAATGTATGAGGCGCTGCCGACCGGACCGGGTAAAGAAGTTGGCACCGTGGTCATTTCCGAAGCCC CGGGCGGGCTGCACTTCAAGGTGAATATGGAAAAGCTGACGCCGGGCTATCATGGCTTTCATGTTCACGA AAATCCAAGCTGCGCTCCGGGAGAAAAAGACGGCAAGATCGTACCGGCTCTTGCTGCCGGCGGGCATTAT GATCCGGGTAATACCCATCACCATTTAGGACCTGAAGGTGATGGACATATGGGCGATTTGCCACGCCTGA GCGCCAATGCTGACGGCAAGGTGAGTGAAACCGTTGTCGCTCCACATCTCAAGAAATTGGCGGAAATCAA GCAGCGTTCTTTGATGGTCCATGTCGGAGGGGATAATTATTCCGATAAGCCTGAGCCGCTTGGTGGCGGT GGTGCCCGTTTTGCCTGCGGCGTGATCGAATA >WP_002972093.1 MULTISPECIES: superoxide dismutase [Cu-Zn] [Brucella] MKSLFIASTMVLMAFPAFAESTTVKMYEALPTGPGKEVGTVVISEAPGGLHFKVNMEKLTPGYHGFHVHE NPSCAPGEKDGKIVPALAAGGHYDPGNTHHHLGPEGDGHMGDLPRLSANADGKVSETVVAPHLKKLAEIK QRSLMVHVGGDNYSDKPEPLGGGGARFACGVIE Protective antigen IMMUNOGENICITY: Induces antigen-specific Th1 immune response, as indicated by the specific induction of serum IgG2a, but not IgG1, antibodies and by the secretion of IFN-γ, but not IL-4, by the Cu/Zn SOD-stimulated splenocytes. Has been used for vaccine development [Ref578:Vemulapalli et al., 2000][Ref579:He et al., 2002][Ref580:Onate et al., 2003]. MUTATION: An isogenic sodC mutant constructed from B abortus 2308 by gene replacement exhibited much greater susceptibility to killing by exogenous O(2)(-) than the parental 2308 strain, supporting a role for SodC in protecting this bacterium from O(2)(-) stress. The B abortus sodC mutant was much more sensitive to killing by cultured resident peritoneal macrophages from C57BL6J mice than 2308, and its attenuation in cultured murine macrophages was enhanced when these phagocytes were treated with gamma interferon. The attenuation of the B abortus sodC mutant in both resting and IFN-gamma-activated macrophages was alleviated in the presence of the NADPH oxidase inhibitor apocynin. Consistently, the B abortus sodC mutant also displayed significant attenuation in infected C57BL6J mice compared to the parental strain. These findings suggest that SodC protects B abortus 2308 from the respiratory burst of host macrophages [Ref581:Gee et al., 2005]. SurA Brucella abortus 2308 VO_0010971 3787419 489056605 BAB_RS19295 JMRZ01000012 WP_002966738 359391 I 688353 689309 - molecular chaperone SurA 10.47 32511.89 318 >NC_007618.1:688353-689309 Brucella melitensis biovar Abortus 2308 chromosome I, complete sequence, strain 2308 GTCAACGATTGACGATGGTGGCCTTTTCGCGCAGTTCCTGCACGTATTTCTTGCTGAGTTCCTCAGCCTT CTTTTCCTGCCCCGCAGGGGAATCCGCGCCTTCCATGGAGAAGACAAGCTGTGCAACGCGGTCATCCGAC ACCTGACGGGTGGAGCATACGGCCAGGAATTCAACACCCTTTTCCGTATCGTGCGGCTTGGTCGTCCGGT TGACGCCAGCGGCCTTCACATCCTTGGACCATTCGCCGGGCAATTGCGGCTCGATGATACGGCCAAGATC GCGGACGGTCACATCGAGAATGCCCTTGGCTTGCTGGCGCGTGCTGTCACAATTCTGGAAGCGCGCGCGC AACGCATTGGCTTCCTGACGGCGTTTTGCCAGAAGAGCGGGCGAACGCTTGGAGGCGGGGACCACGAAAA TCACCTGCTGGAGATGATATTCAGTTGCAACGGGCTTCTTTCCGCCGTTCTTCAGCATACGCTGCACGGC TTCCTGCTCACTGACCATGCCGGTCGCGCGGAAGCGGGCGCTGACAAGGCGGCCCCAGCCCATCTGGACC ATGATATATTTCTTGAAATGTTCCGGCGTGACGCCCGACTGGTTCATAACCTGATTGAGCTGCGCCAGCG TCATCTTGTTGCGGCTTGCAAACCCGGCATAGGCGTCATCCACTTCCTTATCGGAAATATTGATGCCACG CGATTTCATCTCGATGCGCTTGAGCATTTCTTCGGTAAGCTCGTTGCGCGCAAGCTGGTTGAGATTGCCG CTTTTACGCTGAAGCTTGAGGAAGGCCATACGGTGCTTGATGTCGCTATTGGTAATAGCATTGCCGGACA CGATGACTTTGACCTCGGATTCTCCTGCGGCGAAAGCTGGAGCCGCAACCGTTCCAAGCGCCGTCAAGCA AACGGCAGCGCCTAGCATGGAGGCGATAAGAGGTCTTGCAAACATCA >WP_002966738.1 MULTISPECIES: peptidylprolyl isomerase [Brucella] MMFARPLIASMLGAAVCLTALGTVAAPAFAAGESEVKVIVSGNAITNSDIKHRMAFLKLQRKSGNLNQLA RNELTEEMLKRIEMKSRGINISDKEVDDAYAGFASRNKMTLAQLNQVMNQSGVTPEHFKKYIMVQMGWGR LVSARFRATGMVSEQEAVQRMLKNGGKKPVATEYHLQQVIFVVPASKRSPALLAKRRQEANALRARFQNC DSTRQQAKGILDVTVRDLGRIIEPQLPGEWSKDVKAAGVNRTTKPHDTEKGVEFLAVCSTRQVSDDRVAQ LVFSMEGADSPAGQEKKAEELSKKYVQELREKATIVNR Protective antigen Brucella SurA is a protective antigen [Ref687:Delpino et al., 2007b]. Tig Brucella melitensis bv. 1 str. 16M VO_0010969 1196780 17987352 BMEI1069 AE008917 NP_539986 224914 I 1112655 1114112 - trigger factor 4.64 50680.09 485 Tig; RopA; peptidyl-prolyl cis/trans isomerase; promotes folding of newly synthesized proteins; binds ribosomal 50S subunit; forms a homodimer Also called Tf: Trigger Factor. >NC_003317.1:1112655-1114112 Brucella melitensis bv. 1 str. 16M chromosome I, complete sequence ATCAAGCCTCTTCGGACTTGCCTTCTTCGGCCTTCTTCTTCGGCGCAGCCTTCTTCTTCGCAGCCGCCTT CTTGGCAGGCTTTGCTTCCGAAGCTGCATCTTCGTCTTCAGCCGTCAGCTCTTCCTTCGAGACCTTCTTG TCGGTCACATTGATGTTGGCCAGCAGATGATCGACGACCTTTTCTTCAAAGATCGGCGCGCGCAGATTGG CGACGGCATCCGGCGTACGGCGCAGGAAGTCGTAGATTTCCTTTTCCTGACCCGGATAGCGGCGAACCTG ATCGTAAACAGCGCGCTGCAGTTCTTCTTCGGTCACTTCCACGCCTGCCTTCTCGCCGATTTCGGAGAGA ACGAGGCCAAGACGAACGCGGCGTTCTGCAAGCTTGCGATATTCTTCGCGAGCAGCTTCTTCCGTCGTTT CCTCGTCTTCAAAGGTGCGGCCAGCCTGCTGCAGGTCGAAGTTGATCTGCTGCCAGATGTTGTTGAACTC GGCGTCAACCAGCTTCTGCGGGGTTTCAAACTGATAATCACCATCAAGGGCGTCGAGAATCTGACGCTTC ACTTTCTGGCGGGTGATCTGGCCGTACTGGCTTTCGATCTGTTCGCGCACAACCTGACGCAGACGCTCAA GCGACTCAATGCCGAGCTTCTTTGCGGTTTCGTCATCGAGAACGAGTTCGTTCGGCTTGGCAACTTCCTT CACCTTGATGTCGAAGGTTGCTTCCTTGCCAGCCAGATGCGCAGCGCCGTATTCAGCCGGGAACGTTACG GTGATGACCTTCTCGTCGCCAGCCTTCAAGCCAATGAGCTGTTCTTCAAAGCCCGGAATGAACTGGCCGG AACCGAGAACGAGCTGTGCGTCATTGTCTGCACCGCCTTCAAACGGCTCGCCGTCGAGCTTGCCCAGATA GTCGATCGTGACGCGATCTTCGTTTTCGGCCTTGCCCTTCTTGGTTTCAAAGGTGCGGGTCGACGACGCA ATGCGCTTGACCTGTTCATCGACTTCCTCATCGGAAATGTCGACGACTTCACGGGTCACGGCGATCTTGG AGAAGTCCTTGACCTCGATTGCCGGCAGCACTTCATAGTTCAGCGAGAAAACGAAATCGGCCTTGCCGTC GAGAACCTTCTCGGCTTCTTTTTCGTCTTCCGACATGATGACTTCAGGCTGGGTCGCCGACTTTTCGTTG CGTTCGGCAAGGATGGAACGCGACGAATCGTTGAGGATTTCGTTGACGATCTCGGCCATGAAGGACTTGC CGTACATCTTGCGCAGGTGAGCCGTCGGCACCTTGCCCGGACGGAAGCCGTTGATGCGGGCGCGGCCGCG CGCGGTTTCGAGCCGCTCAGCGAGCTTGGCTTCAAGATCCCCGGCCGGAACCACGACTTTGATCTCGCGC TTCAGCCCTTCATTGAGCGTTTCGGTAACCTGCATGTTCAAACCTTCACTTCTTGTCA >NP_539986.1 trigger factor [Brucella melitensis bv. 1 str. 16M] MTRSEGLNMQVTETLNEGLKREIKVVVPAGDLEAKLAERLETARGRARINGFRPGKVPTAHLRKMYGKSF MAEIVNEILNDSSRSILAERNEKSATQPEVIMSEDEKEAEKVLDGKADFVFSLNYEVLPAIEVKDFSKIA VTREVVDISDEEVDEQVKRIASSTRTFETKKGKAENEDRVTIDYLGKLDGEPFEGGADNDAQLVLGSGQF IPGFEEQLIGLKAGDEKVITVTFPAEYGAAHLAGKEATFDIKVKEVAKPNELVLDDETAKKLGIESLERL RQVVREQIESQYGQITRQKVKRQILDALDGDYQFETPQKLVDAEFNNIWQQINFDLQQAGRTFEDEETTE EAAREEYRKLAERRVRLGLVLSEIGEKAGVEVTEEELQRAVYDQVRRYPGQEKEIYDFLRRTPDAVANLR APIFEEKVVDHLLANINVTDKKVSKEELTAEDEDAASEAKPAKKAAAKKKAAPKKKAEEGKSEEA Protective antigen protective antigen [Ref695:Yang et al., 2007] virB2 Brucella melitensis biovar Abortus 2308 3827981 83269021 BAB2_0067 AM040265 YP_418312 359391 II 65610 65927 - type IV secretion system protein VirB2 10.63 10115.27 105 >gi|83268957:65610-65927 Brucella melitensis biovar Abortus 2308 chromosome chromosome II, complete sequence TTTACCTAAGCAGGTAAGAGGCAATTTCGGCGGCAGCGCCAACCACCAGGGCGCCGCCCAGCACCGGCAC TACATCCATGAAGCGGGCGTGCCGGAATGCCATCTTGTAACCGGACCAGATGATGGCTATGGTAACGATG GTGATCGATACGCCGCTTAGCAAGTCCAGCACTTTTTGCATGCTTGTATTTACCTTATCGAGGCCACCGT TGGCGTGCGCCAGGTTAGGCTCGATTGCAGCGATGGAGACAATGAGGGCCAGCAGTAGGTGAGGTAGAAT CCGCGACAGCGACTTCTTGCTGGGGGAAGCGGTTTTCA >gi|83269021|ref|YP_418312.1| type IV secretion system protein VirB2 [Brucella melitensis biovar Abortus 2308] MKTASPSKKSLSRILPHLLLALIVSIAAIEPNLAHANGGLDKVNTSMQKVLDLLSGVSITIVTIAIIWSG YKMAFRHARFMDVVPVLGGALVVGAAAEIASYLLR Virmugen A virB2 mutant is attenuated in Brucella abortus and provides protection in mice [Ref694:Kahl-McDonagh et al., 2007]. vjbR Brucella abortus S19 6326704 189022339 BAbS19_II01090 CP000888 YP_001932080 430066 2 117845 118624 + LuxR family regulatory protein; VjbR 6.76 26640 259 >gi|189022234:117845-118624 Brucella abortus S19 chromosome 2, complete sequence AATGAGTCTTGATCTCGTTCATTTTCCGAATTACAAAAAAACCTTTTTTGGTTCTTCCTTTCAAAGCGAT ACTTTGGCGCTTCTAACCCGCATCCGGGATGAAATAGGATGCCGCTACGTAACGCATACCTATCGTGGTC GCGTAGGCGACTGCACGAAGGTGAATTCAGCCGATCTGACTGTTCTTATGACGCTGCCTGCTACCTGGGT CGCGCGCTATTCCTCGAAAAACTACTTTGCCATTGACCCGGTTTTTCAGGAAGACGCTCCGTATTACCGC AATGATACGAGCGCCATTGCCCGCGACCTGAAAGAAGATGCGGATATTTGCCCGGCCGTTGCCGAACTGT TGCATGATGCTGAAAAGCACGGCCTGGGAAATCTTTTCATCGCCGTATCCGCGCGCAATCCCAAGGGCGT TGCGGGTTGTACGGTTTTCACTTTCGAGGTGGAGGACGAAGACCGTACGCAATTCCTTGCCAGGATGCGC CCGCGCCTTCTAAGCCTTGCCGGCATCATTCATGGAACTGTCTGCGGCTGCAAGGATGCAAATTCAGTTG CAAGCCTTCTCACCCCGCGCGAGGTCGATTGCCTGCGCTGGGCTGCCAATGGCAAGACTGATGGCGAAAT CGCCGAAATCCTCAGCATCGCCCGCTGGACGGTGGTGACTTATCTACAGAATGCCAAAATCAAGCTGAAC TGTTCCAATCGAACGTCGGCAGTGGCGACGGCGCTTTCCCTCGGCATTATCGATATGCCCGAGGTACAGC ATCTCGTCTG >gi|189022339|ref|YP_001932080.1| LuxR family regulatory protein [Brucella abortus S19] MSLDLVHFPNYKKTFFGSSFQSDTLALLTRIRDEIGCRYVTHTYRGRVGDCTKVNSADLTVLMTLPATWV ARYSSKNYFAIDPVFQEDAPYYRNDTSAIARDLKEDADICPAVAELLHDAEKHGLGNLFIAVSARNPKGV AGCTVFTFEVEDEDRTQFLARMRPRLLSLAGIIHGTVCGCKDANSVASLLTPREVDCLRWAANGKTDGEI AEILSIARWTVVTYLQNAKIKLNCSNRTSAVATALSLGIIDMPEVQHLV Virmugen A vjbR mutant is attenuated in BALB/c mice and induces significant protection against wild type challenge [Ref2096:Arenas-Gamboa et al., 2009]. VjbR Brucella melitensis 16M 1198888 17989461 BMEII1116 NC_003318 NP_542094 224914 II 1150819 1151526 - TRANSCRIPTIONAL ACTIVATOR, LUXR FAMILY 6.65 23978.21 235 >gi|1198888|gb|N56040.1|N56040 J6249F Human fetal heart, Lambda ZAP Express Homo sapiens cDNA clone J6249 5', mRNA sequence GTGAACATTTTAGACACCTTTTCTTTGGGTAGGCTCTTGCCCAGGCGCCGGCTCTTCTCCCAAAAAAAAA AAAAACCTTATCCTTTCCCTAACCTGATAACCTCCAGATCCAGGGTTTCTAACTAAGTGAGTCTTTGAAG TATTAAGGGACATGAATAGCT >gi|17989461|ref|NP_542094.1| TRANSCRIPTIONAL ACTIVATOR, LUXR FAMILY [Brucella melitensis 16M] MALLTRIRDEIGCRYVTHTYRGRVGDCTKVNSADLTVLMTLPATWVARYSSKNYFAIDPVFQEDAPYYRN DTSAIARDLKEDADICPAVAELLHDAEKHGLGNLFIAVSARNPKGVAGCTVFTFEVEDEDRTQFLARMRP RLLSLAGIIHGTVCGCKDANSVASLLTPREVDCLRWAANGKTDGEIAEILSIARWTVVTYLQNAKIKLNC SNRTSAVATALSLGIIDMPEVQHLV Virmugen The deletion of vjbR gene in Brucella melitensis strain 16M made a live attenuated vaccine and provided significance protection against mice [Ref1910:Wang et al., 2011]. znuA Brucella melitensis biovar Abortus 2308 VO_0012365 3827700 83269918 BAB2_1079 AM040265 YP_419209 359391 II 1063213 1064217 - periplasmic solute binding protein 6.31 33961.81 334 similar to BRA1122, zinc ABC transporter, periplasmic zinc-bnding protein >gi|83268957:1063213-1064217 Brucella melitensis biovar Abortus 2308 chromosome chromosome II, complete sequence TTTATTTCGGCAGGCAGTCTTTCAGCGAATTGGCCAGATTGCGGATGAGTTGCGGATAAAGATCAGGGCC GTCCTTCAGTTCTGCGCCAAGAGGATCAAGAACACCTGTCCGGGCTTTCGTTCCGTCAACCACCGTCTTC ACCAGTTTCGGCTCAAATTGCGGCTCTGAGAAAACGCAGGTTGCACCGAGTGACTTTATCTTGTCGTGAA TTTGCTGGATACGCGCCGCACCCGGCGCTTTTTCCGGGCTGACCGTGATGGAGCCGGCTGCTTTCATGCC GAAGCGATTCTCGAAATATTGATAGGCATCATGAAAGACGATGAAGGGCTTGTCCTTGACCGGCTTCAGT TCAGCAGCCACTTCCCTGGTCAAGGCATCGAGTTTTTCTCCATAGGCCTTGGCATTCTTTTCATATTGCG CGGCGTGTTCCGGGTCGCTCTCGCCAAGCGTTTTGGCGATATCGGCAGCCAGAATCTTGCCATTTTGCGG GTCAAGCCAGAAATGCAGATCATATTCGCCGTGATGGTGGTGTTCATGGCCTTCTTCTGCGACTTCGGCC GCATGGTCGTGATCGCCTGAACCATGTGCGTCATGCTCTTCCTCATGGCTCCCCCCATGGCCATGATCGT GCGCCTCGAACGGCCCACCCTCGCGGAATTTTAGTTTCGTCAACCCTTTCGCATCGCCAAGAGCCACCAC TTTCGCGCCCTCGCCCAGCGTATCAATGGGCTTGTCGAGAAAGGTTTCCATGGATGGCCCGGCCCAGAAA ATCACCTTGGCGTGCTCGATAGCCTCGGCATCGGAGGGTTTCAGACTATAAACATGCTCGGACCCAGCCC CCTGAACGATCAGCTTCGGCTTACCCACGCCCTGCATGACGGCGGAAACAATCGAGTGAAGCGGCTTGAT CGAAACAACCACGCCCTCACGTTCGCCTGCAAGGGAAGAACCGCAAAAGCCAGCCAGAAAGGCAGAAGCA AGAAACAAGGAATGCAGATTTTTCA >gi|83269918|ref|YP_419209.1| periplasmic solute binding protein [Brucella melitensis biovar Abortus 2308] MKNLHSLFLASAFLAGFCGSSLAGEREGVVVSIKPLHSIVSAVMQGVGKPKLIVQGAGSEHVYSLKPSDA EAIEHAKVIFWAGPSMETFLDKPIDTLGEGAKVVALGDAKGLTKLKFREGGPFEAHDHGHGGSHEEEHDA HGSGDHDHAAEVAEEGHEHHHHGEYDLHFWLDPQNGKILAADIAKTLGESDPEHAAQYEKNAKAYGEKLD ALTREVAAELKPVKDKPFIVFHDAYQYFENRFGMKAAGSITVSPEKAPGAARIQQIHDKIKSLGATCVFS EPQFEPKLVKTVVDGTKARTGVLDPLGAELKDGPDLYPQLIRNLANSLKDCLPK Virmugen Virmugen Deletion of gene znuA attenuates Brucella abortus and this live attenuated vaccine was challenged in mice. 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