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Gary A Splitter

Publications and source records attributed to Gary A Splitter.

At least 19 recordsLinked to original sources

DNA vaccine construct incorporating intercellular trafficking and intracellular targeting motifs effectively primes and induces memory B- and T-cell responses in outbred animals.

We developed a vaccine construct in which a BVP22 domain and an invariant-chain major histocompatibility complex class II-targeting motif capable of enhancing dendritic cell antigen uptake and presentation were fused to a sequence encoding a B- and T-cell antigen from the Anaplasma marginale major surface protein 1a and tested whether this construct would prime and expand immune responses in outbred calves. A single inoculation with this construct effectively primed the immune responses, as demonstrated by a significant enhancement of CD4(+) T-cell proliferation compared to that in calves identically inoculated but inoculated with a DNA construct lacking the targeting domains and compared to that in calves inoculated with an empty vector. These proliferative responses were mirrored by priming and expansion of gamma interferon-positive CD4(+) T cells and immunoglobulin G responses against the linked B-cell epitope. Priming by the single immunization induced memory that underwent rapid recall following reexposure to the antigen. These results demonstrate that DNA vaccines targeting key intercellular and intracellular events significantly enhance priming and expansion and support the feasibility of single-dose DNA immunization in outbred populations.

Animals↗

CGHScan: finding variable regions using high-density microarray comparative genomic hybridization data.

BACKGROUND: Comparative genomic hybridization can rapidly identify chromosomal regions that vary between organisms and tissues. This technique has been applied to detecting differences between normal and cancerous tissues in eukaryotes as well as genomic variability in microbial strains and species. The density of oligonucleotide probes available on current microarray platforms is particularly well-suited for comparisons of organisms with smaller genomes like bacteria and yeast where an entire genome can be assayed on a single microarray with high resolution. Available methods for analyzing these experiments typically confine analyses to data from pre-defined annotated genome features, such as entire genes. Many of these methods are ill suited for datasets with the number of measurements typical of high-density microarrays. RESULTS: We present an algorithm for analyzing microarray hybridization data to aid identification of regions that vary between an unsequenced genome and a sequenced reference genome. The program, CGHScan, uses an iterative random walk approach integrating multi-layered significance testing to detect these regions from comparative genomic hybridization data. The algorithm tolerates a high level of noise in measurements of individual probe intensities and is relatively insensitive to the choice of method for normalizing probe intensity values and identifying probes that differ between samples. When applied to comparative genomic hybridization data from a published experiment, CGHScan identified eight of nine known deletions in a Brucella ovis strain as compared to Brucella melitensis. The same result was obtained using two different normalization methods and two different scores to classify data for individual probes as representing conserved or variable genomic regions. The undetected region is a small (58 base pair) deletion that is below the resolution of CGHScan given the array design employed in the study. CONCLUSION: CGHScan is an effective tool for analyzing comparative genomic hybridization data from high-density microarrays. The algorithm is capable of accurately identifying known variable regions and is tolerant of high noise and varying methods of data preprocessing. Statistical analysis is used to define each variable region providing a robust and reliable method for rapid identification of genomic differences independent of annotated gene boundaries.

Algorithms↗

Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice.

In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B. melitensis mutants, GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091), and the dynamics of bioluminescent virulent bacterial infection following vaccination with these mutants. The virB4, galE, and BMEI1090-BMEI1091 mutants were attenuated in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice; however, only the GR019 (virB4) mutant was attenuated in cultured macrophages. Therefore, in vivo imaging provides a comprehensive approach to identify virulence genes that are relevant to in vivo pathogenesis. Our results provide greater insights into the role of galE in virulence and also suggest that BMEI1090 and downstream genes constitute a novel set of genes involved in Brucella virulence. Survival of the vaccine strain in the host for a critical period is important for effective Brucella vaccines. The galE mutant induced no changes in liver and spleen but localized chronically in the tail and protected IRF-1(-/-) and wild-type mice from virulent challenge, implying that this mutant may serve as a potential vaccine candidate in future studies and that the direct visualization of Brucella may provide insight into selection of improved vaccine candidates.

Animals↗

Enhancement of antigen acquisition by dendritic cells and MHC class II-restricted epitope presentation to CD4+ T cells using VP22 DNA vaccine vectors that promote intercellular spreading following initial transfection.

Induction of immune responses against microbial antigens using DNA is an attractive strategy to mimic the immunity induced by live vaccines. Although DNA vaccines are efficacious in murine models, the requirement for multiple immunizations using high doses in outbred animals and humans has hindered deployment. This requirement is, in part, a result of poor vaccine spreading and suboptimal DC transfection efficiency. Incorporation of a signal that directs intercellular spreading of a DNA-encoded antigen is proposed to mimic live vaccine spreading and increase dendritic cell (DC) presentation. Bovine herpes virus 1 tegument protein, BVP22, is capable of trafficking to surrounding cells. To test the hypothesis that BVP22 enhances spreading and antigen presentation to CD4+ T cells, a DNA construct containing BVP22, fused in-frame to a sequence encoding a T cell epitope of Anaplasma marginale, was generated. A construct with reversed BVP22 sequence served as a negative control. Immunocytometric analysis of transfected primary keratinocytes, human embryonic kidney 293, COS-7, and Chinese hamster ovary cells showed that BVP22 enhanced intercellular spreading by > or = 150-fold. Flow cytometric analysis of antigen-presenting cells (APCs) positively selected from cocultures of transfected cells and APCs showed that 5% of test APCs were antigen-positive, compared with 0.6% of control APCs. Antigen-specific CD4+ T cell proliferation demonstrated that BVP22 enhanced DC antigen presentation by > or = 20-fold. This first report of the ability of BVP22 to increase DNA-encoded antigen acquisition by DCs and macrophages, with subsequent enhancement of major histocompatibility complex class II-restricted CD4+ T cell responses, supports incorporating a spreading motif in a DNA vaccine to target CD4+ T cell-dependent immunity in outbred animals.

Anaplasma marginale↗

Nuclear and mitochondrial localization signals overlap within bovine herpesvirus 1 tegument protein VP22.

VP22, a tegument protein of bovine herpesvirus 1, accumulates in the nucleus of infected and transiently transfected cells. Previous studies indicated a possible regulatory function of VP22 within nuclei, but how VP22 enters nuclei is unknown. Despite the abundance of basic residues within this protein, no classic nuclear localization signal (NLS) motif has been identified. To identify the signal directing nuclear accumulation, a series of truncations, internal deletions, and point mutations were constructed. Fluorescence microscopy of cells transfected with VP22 constructs indicated that a sequence of 103 residues is necessary and sufficient for nuclear localization. This NLS sequence is conformation-sensitive in contrast to a classical sequential NLS. Energy depletion assays and co-immunoprecipitation suggested that this NLS sequence also binds histone H4, resulting in nuclear retention of VP22. In addition, a mitochondrial targeting sequence was identified at the C-terminal 49 amino acids, which overlapped the sequence required for nuclear targeting. Our findings demonstrate the diversity of VP22 protein to localize within the cell and provide the opportunity for VP22 to direct cargo specifically to different subcellular compartments.

Animals↗

Bovine herpesvirus VP22 induces apoptosis in neuroblastoma cells by upregulating the expression ratio of Bax to Bcl-2.

Herpesvirus tegument protein VP22 has been shown to have biotherapeutic potential in tumor gene therapy. Some studies indicate that VP22 may enhance the transfer efficiency of therapeutic proteins by delivering them to more cells while trafficking. Our previous study showed that bovine herpesvirus VP22 (BVP22) enhanced equine herpesvirus thymidine kinase-ganciclovir (Etk-GCV) suicide gene therapy by an unknown intracellular effect. In this study, the interaction between BVP22 and host tumor cells was studied in neuroblastoma NXS2 cells. Cell cycle analysis was performed to determine whether BVP22 possesses biotherapeutic potential by altering the cell cycle, making cells more sensitive to therapeutic genes. As a result, the cell cycle was not affected by the transfection of BVP22 into NXS2 cells. However, cytotoxicity induced by BVP22 was observed in NXS2 cells on the second and third days after transient transfection. Further, analyses of caspase-3 activity and apoptosis suggested that BVP22 induces apoptosis in host tumor cells by upregulating the expression ratio of Bax to Bcl-2.

Animals↗

Temporal analysis of pathogenic events in virulent and avirulent Brucella melitensis infections.

Despite progress in mouse models of brucellosis, much remains unknown regarding Brucella dissemination and tissue localization. Here, we report the dynamics of Brucella infection in individual mice using bioluminescent Brucella melitensis. Bioluminescent imaging of infected interferon regulatory factor-1 knockout (IRF-1(-/-)) mice identified acute infection in many tissues. Brucella was found to replicate in the salivary glands of IRF-1(-/-) and wild-type C57BL/6 mice suggesting a previously unknown tissue preference. Establishing a niche in this region may have relevance in humans where infection can result from ingestion of few bacteria. Sublethal infection of IRF-1(-/-) mice resulted in chronic Brucella localization in tail joints, an infection parallel to osteoarticular brucellosis in humans. Importantly, bioluminescent imaging rapidly identified attenuated EZ::TN/lux mutants in infected mice and revealed differences in dissemination, thereby defining the contribution of Brucella genes to virulence and tissue localization. Surprisingly, a virB mutant, though defective in persistence, disseminated similarly to virulent Brucella, suggesting bacterial spread is independent of VirB proteins that are important for intracellular survival. Together, our results reveal kinetics of acute and chronic Brucella infection in individual mice that parallels human infection as well as readily identified attenuated bacteria. Our approach facilitates identifying virulence determinants that may control tissue specific replication and may help develop therapeutics to overcome Brucella-induced chronic debilitating conditions.

Animals↗

Comparison of bovine leukemia virus (BLV) and CMV promoter-driven reporter gene expression in BLV-infected and non-infected cells.

BACKGROUND: Viral promoters are used in mammalian expression vectors because they generally have strong activity in a wide variety of cells of differing tissues and species. METHODS: The utility of the BLV LTR/promoter (BLVp) for use in mammalian expression vectors was investigated through direct comparison to the CMV promoter (CMVp). Promoter activity was measured using luciferase assays of cell lines from different tissues and species stably transduced with BLVp or CMVp driven luciferase vectors including D17, FLK, BL3.1 and primary bovine B cells. Cells were also modified through the addition of BLV Tax expression vectors and/or BLV infection as well as treatment with trichostatin A (TSA). RESULTS: Results indicate the BLV promoter, while having low basal activity compared to the CMV promoter, can be induced to high-levels of activity similar to the CMV promoter in all cells tested. Tax or BLV infection specifically enhanced BLVp activity with no effect on CMVp activity. In contrast, the non-specific activator, TSA, enhanced both BLVp and CMVp activity. CONCLUSION: Based on these data, we conclude the BLV promoter could be very useful for transgene expression in mammalian expression vectors.

Journal Article↗

The role of integrase/recombinase xerD and monofunctional biosynthesis peptidoglycan transglycosylase genes in the pathogenicity of Brucella abortus infection in vitro and in vivo.

Brucella abortus clones identified previously using a green fluorescence protein reporter system after 4h macrophage infection provided insight regarding possible genes involved in early host-pathogen interaction. Among identified genes were an integrase/recombinase (xerD) gene involved in cell division, and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) gene that catalyzes the final stages of the peptidoglycan membrane synthesis. Here, we evaluate the in vitro and in vivo survival of B. abortus xerD and mtgA insertional mutants. B. abortus xerD::kan and B. abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308. Also, neither gene was required for B. abortus S2308 replication in RAW 264.7 macrophages. However, experimental evidence using interferon regulatory factor 1 knockout mice, a mouse strain highly susceptible to virulent Brucella, revealed that mice infected with B. abortus xerD::kan or B. abortus mtgA::kan survived longer than mice infected with S2308. Additionally, in immunocompetent BALB/c mice, B. abortus xerD::kan had a significantly lower level of bacterial survival when compared to S2308. Together, these results suggest that B. abortus xerD and mtgA genes play a role during the initial phase of infection in mice.

Animals↗

Role of Toll-like receptor 4 in induction of cell-mediated immunity and resistance to Brucella abortus infection in mice.

Initial host defense to bacterial infection is executed by innate immunity, and therefore the main goal of this study was to examine the contribution of Toll-like receptors (TLRs) during Brucella abortus infection. CHO reporter cell lines transfected with CD14 and TLRs showed that B. abortus triggers both TLR2 and TLR4. In contrast, lipopolysaccharide (LPS) and lipid A derived from Brucella rough (R) and smooth (S) strains activate CHO cells only through TLR4. Consistently, macrophages from C3H/HePas mice exposed to R and S strains and their LPS produced higher levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-12 compared to C3H/HeJ, a TLR4 mutant mouse. The essential role of TLR4 for induction of proinflammatory cytokines was confirmed with diphosphoryl lipid A from Rhodobacter sphaeroides. Furthermore, to determine the contribution of TLR2 and TLR4 in bacterial clearance, numbers of Brucella were monitored in the spleen of C3H/HeJ, C3H/HePas, TLR2 knockout, and wild-type mice at 1, 3, and 6 weeks following B. abortus infection. Interestingly, murine brucellosis was markedly exacerbated at weeks 3 and 6 after infection in animals that lacked functional TLR4 (C3H/HeJ) compared to C3H/HePas that paralleled the reduced gamma interferon production by this mouse strain. Finally, by mass spectrometry analysis we found dramatic differences on the lipid A profiles of R and S strains. In fact, S lipid A was shown to be more active to trigger TLR4 than R lipid A in CHO cells and more effective in inducing dendritic cell maturation. In conclusion, these results indicate that TLR4 plays a role in resistance to B. abortus infection and that S lipid A has potent adjuvant activity.

Animals↗

Comparative whole-genome hybridization reveals genomic islands in Brucella species.

Brucella species are responsible for brucellosis, a worldwide zoonotic disease causing abortion in domestic animals and Malta fever in humans. Based on host preference, the genus is divided into six species. Brucella abortus, B. melitensis, and B. suis are pathogenic to humans, whereas B. ovis and B. neotomae are nonpathogenic to humans and B. canis human infections are rare. Limited genome diversity exists among Brucella species. Comparison of Brucella species whole genomes is, therefore, likely to identify factors responsible for differences in host preference and virulence restriction. To facilitate such studies, we used the complete genome sequence of B. melitensis 16M, the species highly pathogenic to humans, to construct a genomic microarray. Hybridization of labeled genomic DNA from Brucella species to this microarray revealed a total of 217 open reading frames (ORFs) altered in five Brucella species analyzed. These ORFs are often found in clusters (islands) in the 16M genome. Examination of the genomic context of these islands suggests that many are horizontally acquired. Deletions of genetic content identified in Brucella species are conserved in multiple strains of the same species, and genomic islands missing in a given species are often restricted to that particular species. These findings suggest that, whereas the loss or gain of genetic material may be related to the host range and virulence restriction of certain Brucella species for humans, independent mechanisms involving gene inactivation or altered expression of virulence determinants may also contribute to these differences.

Animals↗

Denitrification genes regulate Brucella virulence in mice.

Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. Genome sequencing of B. suis and B. melitensis revealed that both are complete denitrifiers. To learn more about the role of denitrification in these animal pathogens, a study of the role of denitrification in the closely related B. neotomae was undertaken. In contrast to B. suis and B. melitensis, it was found that B. neotomae is a partial denitrifier that can reduce nitrate to nitrite but no further. Examination of the B. neotomae genome showed that a deletion in the denitrification gene cluster resulted in complete loss of nirV and the partial deletion of nirK and nnrA. Even though the nor operon is intact, a norC-lacZ promoter fusion was not expressed in B. neotomae. However, the norC-lacZ fusion was expressed in the related denitrifier Agrobacterium tumefaciens, suggesting that the lack of expression in B. neotomae is due to inactivation of NnrA. A narK-lacZ promoter fusion was found to exhibit nitrate-dependent expression consistent with the partial denitrifier phenotype. Complementation of the deleted region in B. neotomae by using nirK, nirV, and nnrA from B. melitensis restored the ability of B. neotomae to reduce nitrite. There was a significant difference in the death of IRF-1-/- mice when infected with B. neotomae containing nirK, nirV, and nnrA and those infected with wild-type B. neotomae. The wild-type strain killed all the infected mice, whereas most of the mice infected with B. neotomae containing nirK, nirV, and nnrA survived.

Adaptation, Physiological↗

Bovine herpesvirus tegument protein VP22 enhances thymidine kinase/ganciclovir suicide gene therapy for neuroblastomas compared to herpes simplex virus VP22.

Herpesvirus tegument protein VP22 can enhance the effect of therapeutic proteins in gene therapy, such as thymidine kinase (tk) and p53; however, the mechanism is unclear or controversial. In this study, mammalian expression vectors carrying bovine herpesvirus 1 (BHV-1) VP22 (BVP22) or herpes simplex virus type 1 (HSV-1) VP22 (HVP22) and equine herpesvirus type 4 (EHV-4) tk (Etk) were constructed in order to evaluate and compare the therapeutic potentials of BVP22 and HVP22 to enhance Etk/ganciclovir (Etk/GCV) suicide gene therapy for neuroblastomas by GCV cytotoxicity assays and noninvasive bioluminescent imaging in vitro and in vivo. BVP22 enhanced Etk/GCV cytotoxicity compared to that with HVP22 both in vitro and in vivo. However, assays utilizing a mixture of parental and stably transfected cells indicated that the enhancement was detected only in transfected cells. Thus, the therapeutic potential of BVP22 and HVP22 in Etk/GCV suicide gene therapy in this tumor system is not due to VP22 delivery of Etk into surrounding cells but rather is likely due to an enhanced intracellular effect.

Animals↗

Molecular host-pathogen interaction in brucellosis: current understanding and future approaches to vaccine development for mice and humans.

Brucellosis caused by Brucella spp. is a major zoonotic disease. Control of brucellosis in agricultural animals is a prerequisite for the prevention of this disease in human beings. Recently, Brucella melitensis was declared by the Centers for Disease Control and Prevention to be one of three major bioterrorist agents due to the expense required for the treatment of human brucellosis patients. Also, the economic agricultural loss due to bovine brucellosis emphasizes the financial impact of brucellosis in society. Thus, vaccination might efficiently solve this disease. Currently, B. abortus RB51 and B. melitensis REV.1 are used to immunize cattle and to immunize goats and sheep, respectively, in many countries. However, these genetically undefined strains still induce abortion and persistent infection, raising questions of safety and efficiency. In fact, the REV.1 vaccine is quite virulent and apparently unstable, creating the need for improved vaccines for B. melitensis. In addition, Brucella spp. may or may not provide cross-protection against infection by heterologous Brucella species, hampering the acceleration of vaccine development. This review provides our current understanding of Brucella pathogenesis and host immunity for the development of genetically defined efficient vaccine strains. Additionally, conditions required for an effective Brucella vaccine strain as well as the future research direction needed to investigate Brucella pathogenesis and host immunity are postulated.

Animals↗

Susceptibility of IFN regulatory factor-1 and IFN consensus sequence binding protein-deficient mice to brucellosis.

IFN-gamma is a key cytokine controlling Brucella infection, and the diverse functions of this cytokine are mediated by IFN regulatory factors (IRFs) such as IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). However, the roles of these three IRFs in Brucella infection have not been investigated. The infection of each IRF-deficient mouse strain provides an opportunity to determine not only the significance of each IRF molecule but also the crucial immune components necessary for host defense during in vivo infection, because respective IRF-deficient mouse strains contain unique immunodeficient phenotypes. Brucella abortus S2308-infected IRF-1-/- mice were dead within 2 wk postinfection, while IRF-2-/- mice contained less splenic Brucella CFU than wild-type mice at the early stage of infection. Infected ICSBP-/- mice maintained a plateau of splenic Brucella CFU throughout the infection. Additional infection of IL-12p40-, NO synthase 2-, and gp91(phox)-deficient mice indicates that these immune components are crucial for Brucella immunity and may contribute to the susceptibility of IRF-1-/- and ICSBP-/- mice. Immunologic and histopathological analyses of infected IRF-1-/- mice indicate that the absence of IL-12p40 induction and serious hepatic damage are involved in the death of IRF-1-/- mice. These results indicate that 1) IRF-1 and ICSBP are essential transcriptional factors for IFN-gamma-mediated protection against Brucella; 2) IL-12, reactive nitrogen intermediates, and reactive oxygen intermediates are crucial immune components against Brucella, and their absence may contribute to the susceptibility of IRF-1-/- and ICSBP-/- mice; and 3) hepatic damage caused by Brucella virulence contributes to the death of IRF-1-/- mice.

Animals↗

Encephalomyocarditis and Mengo viruses productively infect murine T-lymphocyte cell lines but not fresh ex vivo derived T lymphocytes.

Encephalomyocarditis virus (EMCV) and Mengo virus are highly virulent murine cardioviruses that are found in abundant quantities in the spleen and lymph nodes after infection. T lymphocytes are pivotal mediators of humoral and cellular immunity against cardioviral challenge, and are highly suspect candidates of EMCV and Mengo virus infection. We found T lymphocyte-like cell lines CTLL-2, EL-4, LY1+2/9, and LBRM33 were susceptible to productive viral infection and exhibited cytopathology after infection with virulent EMCV-R or attenuated Mengo virus strains vMC0 and vMC24. Flow cytometric analysis demonstrated progressive intracellular accumulation of viral proteins, such as the replication-dependent 3D viral polymerase, in EL-4 cells during infection. Conversely, freshly isolated and mitogen-stimulated CD4+ and CD8+ T cells were resistant to productive infection with these viruses, exhibiting no viral-induced cytopathic effects or intracellular presence of viral proteins. These data indicate that although T-lymphocyte-like tumor cell lines are highly susceptible to viral infection and cytopathic effects, primary/freshly isolated T cells are resistant to infection by EMCV-R or Mengo virus.

Animals↗

Molecular and immunological characterisation of recombinant Brucella abortus glyceraldehyde-3-phosphate-dehydrogenase, a T- and B-cell reactive protein that induces partial protection when co-administered with an interleukin-12-expressing plasmid in a DNA vaccine formulation.

To identify antigens of Brucella spp. that are potentially involved in stimulating a protective T-cell-mediated immune response, previous studies identified 10 clones from a Brucella abortus 2308 genomic library with primed lymphocytes as probes. One selected positive clone (182) contained an insert of 1.2 kb which was identified, sequenced and characterised. The deduced amino acid sequence of the open reading frame (ORF) revealed 82% and 81% identity to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes from Agrobacterium tumefaciens and Xanthobacter flavus, respectively. Southern blot analysis demonstrated that the gap gene is present in only one copy in the Brucella genome. B. abortus GAPDH was then expressed in Escherichia coli as a fusion protein with the maltose-binding protein (MBP). To demonstrate the functional activity of Brucella GAPDH, E. coli gap mutants were transformed with a Brucella pMAL-gap construct. Genetic complementation was achieved and as a result E. coli mutants were able to grow on glucose or other carbon source medium. The humoral and cellular immune responses to the recombinant (r) GAPDH were characterised. In Western blots, sera from naturally infected cattle and sheep showed antibody reactivity against rGAPDH. 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, gap associated with murine IL-12 gene in a DNA vaccine formulation partially protected mice against experimental infection.

Amino Acid Sequence↗

Virulence criteria for Brucella abortus strains as determined by interferon regulatory factor 1-deficient mice.

Interferon regulatory factor 1-deficient (IRF-1(-/-)) mice infected with virulent Brucella abortus 2308 at 5 x 10(5) CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF-1(-/-) mice were unable to resolve infection and died. In contrast, IRF-1(-/-) mice survived when infected at 5 x 10(5) CFU with several attenuated Brucella strains (S19, RB51, cbp, and cyd). The survival of infected IRF-1(-/-) mice is likely a function of the level of virulence of each Brucella strain and the extent of retained immunity. Further, these findings suggest that adaptive immunity may be important to the survival of IRF-1(-/-) mice since attenuated Brucella strains can protect IRF-1(-/-) mice against lethal challenge with virulent Brucella: Using the IRF-1(-/-) mouse model, the following set of criteria were identified to define Brucella virulence: (i) the day of death for 50% of mice infected with 5 x 10(5)CFU of Brucella, (ii) the extent of liver toxicity, and (iii) the minimum immunizing dose of Brucella to protect against challenge with virulent S2308. Thus, IRF-1(-/-) mice are important to determining the level of Brucella virulence, to evaluating Brucella mutants for attenuation, and to investigating adaptive immunity in brucellosis.

Animals↗