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BackgroundBrucellosis remains a significant health and economic challenge for livestock and humans globally. Despite its public health implications, the factors driving the endemic persistence of Brucella at the human-livestock interface in Tanzania remain poorly elucidated. This study aimed to identify the seroprevalence of Brucella infection in livestock and humans within a ranching system and determine associated risk factors for disease endemicity.MethodsA cross-sectional sero-epidemiological study was conducted in 2023 in Tanzania’s Karagwe District, involving 725 livestock (cattle, goats, sheep) from 10 herds and 112 humans from associated camps. Seroprevalence was assessed using competitive ELISA while epidemiological data were collected via questionnaires. Generalized Linear Models and Contrast Analysis were used to identify risk factors for infection.ResultsOverall seroprevalence was 34% in livestock and 41% in humans. Goats exhibited the highest prevalence (69.2%), while cattle had the lowest (22.6%). Mixed-species herds (Odds Ratio, OR = 2.96, CI [1.90–4.60]) and small ruminants-only herds (OR = 6.54, CI [3.65–11.72]) showed a significantly higher risk of seropositivity compared to cattle-only herds. Older cattle (OR = 5.23, CI [2.70–10.10]) and lactating females (OR = 2.87, CI [1.78–4.63]) represented significant risks for brucellosis in livestock. In humans, close contact with animals (OR = 7.20, CI [1.97–36.31]) and handling animals during parturition or aborted fetuses (OR = 2.37, CI [1.01–5.58]) were significant risk factors. Notably, no spatial association was found in seroprevalence between herds and nearby human communities.ConclusionThe lack of spatial correlation between livestock and human seroprevalence suggests complex transmission dynamics, potentially involving endemic circulation in livestock and human infections from multiple sources of exposure to livestock. This study highlights the need for comprehensive zoonotic risk education and targeted intervention strategies. Further research is crucial to elucidate transmission pathways and improve Brucella infection control. This includes developing robust methods for identifying infective species and implementing effective strategies to mitigate Brucella infection in endemic regions.

Johne’s disease (JD), also known as paratuberculosis, is a chronic, untreatable gastroenteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection. Evidence for host genetic resistance to disease progression exists, although it is limited due to the extended incubation period (years) and diagnostic challenges. To overcome this, previously restored formalin-fixed paraffin embedded tissue (FFPE) DNA from archived FFPE tissue cassettes was utilized for a novel retrospective case-control genome-wide association study (GWAS) on ovine JD. Samples from known MAP-infected flocks with ante- and postmortem diagnostic data were used. Cases (N = 9) had evidence of tissue infection, compared to controls (N = 25) without evidence of tissue infection despite positive antemortem diagnostics. A genome-wide efficient mixed model analysis (GEMMA) to conduct a GWAS using restored FFPE DNA SNP results from the Illumina Ovine SNP50 Bead Chip, identified 10 SNPs reaching genome-wide significance of p < 1 × 10−6 on chromosomes 1, 3, 4, 24, and 26. Pathway analysis using PANTHER and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was completed on 45 genes found within 1 Mb of significant SNPs. Our work provides a framework for the novel use of archived FFPE tissues for animal genetic studies in complex diseases and further evidence for a genetic association in JD.

Probiotics for humans and direct-fed microbials for livestock are increasingly popular dietary ingredients for supporting immunity. The aim of this study was to determine the effects of dietary supplementation of Bacillus subtilis MB40 (MB40) on immunity in piglets challenged with the foodborne pathogen Listeria monocytogenes (LM). Three-week-old piglets (n = 32) were randomly assigned to four groups: (1) basal diet, (2) basal diet with LM challenge, (3) MB40-supplemented diet, and (4) MB40-supplemented diet with LM challenge. Experimental diets were provided throughout a 14-day (d) period. On d8, piglets in groups 2 and 4 were intraperitoneally inoculated with LM at 108 CFU/mL per piglet. Blood samples were collected at d1, d8, and d15 for biochemical and immune response profiling. Animals were euthanized and necropsied at d15 for liver and spleen bacterial counts and intestinal morphological analysis. At d15, LM challenge was associated with increased spleen weight (p = 0.017), greater circulating populations of neutrophils (p = 0.001) and monocytes (p = 0.008), and reduced ileal villus height to crypt depth ratio (p = 0.009), compared to non-challenged controls. MB40 supplementation reduced LM bacterial counts in the liver and spleen by 67% (p < 0.001) and 49% (p < 0.001), respectively, following the LM challenge, compared to the basal diet. MB40 supplementation was also associated with decreased circulating concentrations of monocytes (p = 0.007). Altogether, these data suggest that MB40 supplementation is a safe and well-tolerated approach to enhance immunity during systemic Listeria infection.

Paratuberculosis, or Johne's Disease (JD) is a debilitating chronic enteritis mainly affecting ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). This organism causes worldwide economic losses to the livestock industry, and is of public health importance due to the potential zoonotic risk between MAP and Crohn's disease (CD) in humans. Without economical treatments, or a vaccine capable of preventing infection without causing cross-reactions with bovine tuberculosis, test-and-cull methods for disease control are imperative. Unfortunately, difficulties in diagnostics and long subclinical stage hinder adequate control and is further complicated by variation in MAP exposure outcome. Interestingly, the majority of infections result in asymptomatic presentation and never progress to clinical disease. One contributing factor is host genetics, where polymorphisms in innate immune genes have been found to influence resistance and susceptibility to disease. Candidate genes identified across studies overlap with those found in CD and tuberculosis including; Solute carrier family 11 member 1 gene (SLC11A1), Nucleotide-binding-oligomerization domain containing gene 2 (NOD2), Major histocompatibility complex type II (MHC-II), and Toll-like receptor (TLR) genes. This review will highlight evidence supporting the vital role of these genes in MAP infection outcome, associated challenges, and implications for the future of JD research.

Population and health management of wildlife is a key to environmental health, domestic herd health, and ultimately public health. Many different methods including: surgical sterilization, poison baits, and sponsored hunting programs have been used in the attempt to control populations of various nuisance animal species. Particular interest has been given to immunocontraception through wildlife vaccination protocols. This study specifically looked at the potential immunocontraceptive and protective properties of a Brucella abortus RB51 ΔleuB vaccine expressing Salmonella typhimurium FliC conjugated to porcine follicle stimulating hormone beta subunit (FSHβ) or gonadotropin releasing hormone (GnRH) DNA sequences. B. abortus RB51 ΔleuB pNS4-TrcD-FliC- FSHβ (RB51LFSHβ) and B. abortus RB51 ΔleuB pNS4-TrcD-FliC-GnRH (RB51LGnRH) were tested in a pilot breeding study with BALB/c mice, and a significant reduction in fertility characteristics was observed in both male and female mice. Ultimately, this study provides support to test these vaccine candidates in feral swine, a destructive invasive species in the United States of America.

Membrane blebs are released from Gram-negative bacteria, however, little is known about Brucella blebs. This work pursued two objectives, the first was to determine and identify the proteins in the membrane blebs by proteomics and in silico analysis. The second aim was to evaluate the use of membrane blebs of Brucella abortus 2308 and B. abortus RB51 as an acellular vaccine in vivo and in vitro. To achieve these aims, membrane blebs from B. abortus 2308 and RB51 were obtained and then analyzed by liquid chromatography coupled to mass spectrometry. Brucella membrane blebs were used as a “vaccine” to induce an immune response in BALB/c mice, using the strain B. abortus RB51 as a positive vaccine control. After subsequent challenge with B. abortus 2308, CFUs in spleens were determined; and immunoglobulins IgG1 and IgG2a were measured in murine serum by ELISA. Also, activation and costimulatory molecules induced by membrane blebs were analyzed in splenocytes by flow cytometry. Two hundred and twenty eight proteins were identified in 2308 membrane blebs and 171 in RB51 blebs, some of them are well-known Brucella immunogens such as SodC, Omp2b, Omp2a, Omp10, Omp16, and Omp19. Mice immunized with membrane blebs from rough or smooth B. abortus induced similar protective immune responses as well as the vaccine B. abortus RB51 after the challenge with virulent strain B. abortus 2308 (P < 0.05). The levels of IgG2a in mice vaccinated with 2308 membrane blebs were higher than those vaccinated with RB51 membrane blebs or B. abortus RB51 post-boosting. Moreover, mice immunized with 2308 blebs increased the percentage of activated B cells (CD19+CD69+) in vitro. Therefore, membrane blebs are potential candidates for the development of an acellular vaccine against brucellosis, especially those derived from the rough strains so that serological diagnostic is not affected.

In 1967, Brucella neotomae was first isolated from Neotoma lepida, the dessert wood rat, in Utah. With little infection data since its discovery, the zoonotic potential of this Brucella species is largely unknown. Recent reports of isolation from human cerebrospinal fluid, along with current literature suggest that B. neotomae has the ability to infect various hosts and cell types. In this report we extend the knowledge of B. neotomae ATCC 23459's intracellular invasion and survival abilities to a variety of cell lines through gentamicin protection assays. Some of the phagocytic and epithelial cell lines from various mammalian species represent characteristics of some cell types that could be encountered by Brucella in potential hosts. It was found that B. neotomae ATCC 23459 exhibits generally lower intracellular bacterial CFUs compared to the mouse-passaged strain of B. neotomae ATCC 23459, B. suis 1330, and B. abortus 2308. Ultimately, these observations provide a small piece of the puzzle in the investigation of the breadth of B. neotomae's pathogenic potential.

Brucella abortus RB51 is an attenuated, stable, spontaneous rough mutant derived in the laboratory from the virulent strain B. abortus 2308. Previous studies discovered that the wboA gene, which encodes a glycosyltransferase required for synthesis of the O-polysaccharide, is disrupted in strain RB51 by an IS711 element. However, complementation of strain RB51 with a functional wboA gene (strain RB51WboA) does not confer it a smooth phenotype but results in low levels of cytoplasmic O-polysaccharide synthesis. In this study, we asked if increasing the potential availability of bactoprenol priming precursors in strain RB51WboA would increase the levels of O-polysaccharide synthesis and enhance the protective efficacy against virulent Brucella challenge. To achieve this, we overexpressed the wbkF gene, which encodes a putative undecaprenyl-glycosyltransferase involved in bactoprenol priming for O-polysaccharide polymerization, in strain RB51WboA to generate strain RB51WboAKF. In comparison to strain RB51WboA, strain RB51WboAKF expressed higher levels of O-polysaccharide, but was still attenuated and remained phenotypically rough. Mice immunized with strain RB51WboAKF developed increased levels of smooth LPS-specific serum antibodies, primarily of IgG2a and IgG3 isotype. Splenocytes from mice vaccinated with strain RB51WboAKF secreted higher levels of antigen-specific IFN-γ and TNF-α and contained more numbers of antigen-specific IFN-γ secreting CD4+ and CD8+ T lymphocytes when compared to those of the RB51 or RB51WboA vaccinated groups. Immunization with strain RB51WboAKF conferred enhanced protection against virulent B. abortus 2308, B. melitensis 16M and B. suis 1330 challenge when compared to the currently used vaccine strains. Our results suggest that strain RB51WboAKF has the potential to be a more efficacious vaccine than its parent strain in natural hosts.

Bacteriophage O1 is a Myoviridae A1 group member used historically for identifying Salmonella. Sequencing revealed a single, linear, 86,155-base-pair genome with 39% average G+C content, 131 open reading frames, and 22 tRNAs. Closest protein homologs occur in Erwinia amylovora phage φEa21-4 and Escherichia coli phage wV8. Proteomic analysis indentified structural proteins: Gp23, Gp36 (major tail protein), Gp49, Gp53, Gp54, Gp55, Gp57, Gp58 (major capsid protein), Gp59, Gp63, Gp64, Gp67, Gp68, Gp69, Gp73, Gp74 and Gp77 (tail fiber). Based on phage-host codon differences, 7 tRNAs could affect translation rate during infection. Introns, holin-lysin cassettes, bacterial toxin homologs and host RNA polymerase-modifying genes were absent.

Brucella abortus S19 and RB51 strains have been successfully used to control bovine brucellosis worldwide; however, currently, most of our understanding of the protective immune response induced by vaccination comes from studies in mice. The aim of this study was to characterize and compare the immune responses induced in cattle prime-immunized with B. abortus S19 or RB51 and revaccinated with RB51. Female calves, aged 4 to 8 months, were vaccinated with either vaccine S19 (0.6-1.2 x 1011 CFU) or RB51 (1.3 x 1010 CFU) on day 0, and revaccinated with RB51 (1.3 x 1010 CFU) on day 365 of the experiment. Characterization of the immune response was performed using serum and peripheral blood mononuclear cells. Blood samples were collected on days 0, 28, 210, 365, 393 and 575 post-immunization. Results showed that S19 and RB51 vaccination induced an immune response characterized by proliferation of CD4+ and CD8+ T-cells; IFN-ɣ and IL-17A production by CD4+ T-cells; cytotoxic CD8+ T-cells; IL-6 secretion; CD4+ and CD8+ memory cells; antibodies of IgG1 class; and expression of the phenotypes of activation in T-cells. However, the immune response stimulated by S19 compared to RB51 showed higher persistency of IFN-ɣ and CD4+ memory cells, induction of CD21+ memory cells and higher secretion of IL-6. After RB51 revaccination, the immune response was chiefly characterized by increase in IFN-ɣ expression, proliferation of antigen-specific CD4+ and CD8+ T-cells, cytotoxic CD8+ T-cells and decrease of IL-6 production in both groups. Nevertheless, a different polarization of the immune response, CD4+- or CD8+-dominant, was observed after the booster with RB51 for S19 and RB51 prime-vaccinated animals, respectively. Our results indicate that after prime vaccination both vaccine strains induce a strong and complex Th1 immune response, although after RB51 revaccination the differences between immune profiles induced by prime-vaccination become accentuated.

The plasma membrane contains discrete nanometer-sized domains that are resistant to non-ionic detergents, and which are called detergent resistant membrane domains (DRMDs) or lipid rafts. Exposure of host cells to pathogenic bacteria has been shown to induce the re-distribution of specific host proteins between DRMDs and detergent soluble membranes, which leads to the initiation of cell signaling that enable pathogens to access host cells. DRMDs have been shown to play a role in the invasion of Brucella into host macrophages and the formation of replicative phagosomes called Brucella-containing vacuoles (BCVs). In this study we sought to characterize changes to the protein expression profiles in DRMDs and to respective cellular pathways and networks of Mono Mac 6 cells in response to the adherence of rough VTRM1 and smooth 16 M B. melitensis strains. DRMDs were extracted from Mono Mac 6 cells exposed for 2 minutes at 4°C to Brucella (no infection occurs) and from unexposed control cells. Protein expression was determined using the non-gel based quantitative iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry technique. Using the identified iTRAQ proteins we performed enrichment analyses and probed constructed human biochemical networks for interactions and metabolic reactions. We identified 149 proteins, which either became enriched, depleted or whose amounts did not change in DRMDs upon Brucella exposure. Several of these proteins were distinctly enriched or depleted in DRMDs upon exposure to rough and smooth B. melitensis strains which results in the differential engagement of cellular pathways and networks immediately upon Brucella encounter. For some of the proteins such as myosin 9, small G protein signaling modulator 3, lysine-specific demethylase 5D, erlin-2, and voltage-dependent anion-selective channel protein 2, we observed extreme differential depletion or enrichment in DRMDs. The identified proteins and pathways could provide the basis for novel ways of treating or diagnosing Brucellosis.

Brucella spp. are Gram-negative, facultative intracellular coccobacilli that cause one of the most frequently encountered zoonosis worldwide. Humans naturally acquire infection through consumption of contaminated dairy and meat products and through direct exposure to aborted animal tissues and fluids. No vaccine against brucellosis is available for use in humans. In this study, we tested the ability of orally inoculated gamma-irradiated B. neotomae and B. abortus RB51 in a prime-boost immunization approach to induce antigen-specific humoral and cell mediated immunity and protection against challenge with virulent B. abortus 2308. Heterologous prime-boost vaccination with B. abortus RB51 and B. neotomae and homologous prime-boost vaccination of mice with B. neotomae led to the production of serum and mucosal antibodies specific to the smooth LPS. The elicited serum antibodies included the isotypes of IgM, IgG1, IgG2a, IgG2b and IgG3. All oral vaccination regimens induced antigen-specific CD4(+) and CD8(+) T cells capable of secreting IFN-γ and TNF-α. Upon intra-peritoneal challenge, mice vaccinated with B. neotomae showed the highest level of resistance against virulent B. abortus 2308 colonization in spleen and liver. Experiments with different doses of B. neotomae showed that all tested doses of 10(9), 10(10) and 10(11) CFU-equivalent conferred significant protection against the intra-peritoneal challenge. However, a dose of 10(11) CFU-equivalent of B. neotomae was required for affording protection against intranasal challenge as shown by the reduced bacterial colonization in spleens and lungs. Taken together, these results demonstrate the feasibility of using gamma-irradiated B. neotomae as an effective and safe oral vaccine to induce protection against respiratory and systemic infections with virulent Brucella.

The outer membrane vesicles (OMVs) from smooth B. melitensis 16 M and a derived rough mutant, VTRM1 strain, were purified and characterized with respect to protein content and induction of immune responses in mice. Proteomic analysis showed 29 proteins present in OMVs from B. melitensis 16 M; some of them are well-known Brucella immunogens such as SOD, GroES, Omp31, Omp25, Omp19, bp26, and Omp16. OMVs from a rough VTRM1 induced significantly higher expression of IL-12, TNFα, and IFNγ genes in bone marrow dendritic cells than OMVs from smooth strain 16 M. Relative to saline control group, mice immunized intramuscularly with rough and smooth OMVs were protected from challenge with virulent strain B. melitensis 16 M just as well as the group immunized with live strain B. melitensis Rev1 (P

Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.

Abstract  A rapid and simple method for preparation of highly efficient Ochrobactrum anthropi electrocompetent cells has been developed. The efficiency of transformation increased 200-fold when the cells were prepared from liquid culture compared to agar plates. Effects of different conditions, including cell density, electric field strength, resistance and plasmid size were evaluated to develop an electroporation protocol. The electrocompetent O. anthropi prepared by this method were 9-fold more efficient than commercial sources of competent Escherichia coli. The method described here will enhance the genetic manipulation of Ochrobactrum as a bioremediation tool and a biopesticide agent. [ABSTRACT FROM AUTHOR]