Search Results (13)

Brucellosis in small ruminants, primarily caused by B. melitensis, remains a significant threat to public health in many regions. Although early-age vaccination of breeding stocks was expected to facilitate infection control, this approach did not meet expectations, while in some cases, late vaccination of animals has been associated with an increased number of human cases. Therefore, in this field study, we investigated the immune response and bacteremia cases in ten apparently healthy hoggets vaccinated at the age of nine months. Before vaccination, the hoggets were seronegative and negative in blood cultures, although B. melitensis DNA was detected in three animals using PCR. After vaccination, twelve Brucella spp. strains were isolated from the blood cultures of nine hoggets at different time points. Whole genome analysis identified eleven of them as identical to three B. melitensis strains previously isolated in Greece. The tested animals completed their gestation without any adverse outcomes. According to our results, late vaccination, despite extending animal exposure to B. melitensis, apparently protects against disease and abortion but not against infection. The onset of post-vaccination immune response may be influenced by transient infections by field strains. Full article

Brucellosis is a neglected zoonotic disease affecting livestock and humans that remains endemic in Ethiopia. Despite its prevalence, only a few studies have identified Brucella species circulating in livestock in the country. This study aimed to determine the Brucella species responsible for infections in livestock in the Afar region of Ethiopia and characterize the isolates using whole-genome single nucleotide polymorphism (wgSNP) analysis and in silico multi-locus sequence typing (MLST). Comparisons were made between Ethiopian Brucella and regional and global isolates to determine their phylogenetic relationships. Surveys conducted in May and October–November 2022 in six villages of the Amibara district involved the collection of vaginal swabs (n = 231) and milk samples (n = 17) from 32 sheep and 199 goats kept by 143 pastoral households reporting recent abortions in the animals. Brucella melitensis was detected in three sheep and 32 goats, i.e., 15% (35/231) of animals across 20% (29/143) of households using bacterial culture and PCR-based methods (bcsp31, AMOS, and Bruce-ladder multiplex PCR). Of the 35 positive animals, B. melitensis was isolated from 24 swabs, while the remaining 11 were culture-negative and detected only by PCR. The genomic DNA of the 24 isolates was sequenced using Illumina Novaseq 6000 and assembled using the SPAdes pipeline. Nine- and 21-locus MLST identified 23 isolates as genotype ST12, while one isolate could not be typed. The wgSNP-based phylogenetic analysis revealed that the Ethiopian isolates clustered within the African clade and were closely related to isolates from Somalia. Several virulence factors responsible for adhesion, intracellular survival, and regulatory functions were detected in all isolates. No antimicrobial resistance genes associated with resistance to drugs commonly used for treating brucellosis were detected. Since B. melitensis is prevalent in sheep and goats, vaccination with the B. melitensis Rev-1 vaccine is the recommended strategy in these pastoral systems to protect animal and human health. Full article

Brucellosis is a bacterial zoonosis caused by the genus Brucella, which mainly affects domestic animals. In these natural hosts, brucellae display a tropism towards the reproductive organs, such as the placenta, replicating in high numbers and leading to placentitis and abortion, an ability also exerted by the B. melitensis live-attenuated Rev1 strain, the only vaccine available for ovine brucellosis. It is broadly accepted that this tropism is mediated, at least in part, by the presence of certain preferred nutrients in the placenta, particularly erythritol, a polyol that is ultimately incorporated into the Brucella central carbon metabolism via two reactions dependent on transaldolase (Tal) or fructose-bisphosphate aldolase (Fba). In the light of these remarks, we propose that blocking the incorporation of erythritol into the central carbon metabolism of Rev1 by deleting the genes encoding Tal and Fba may impair the ability of the vaccine to proliferate massively in the placenta. Therefore, a Rev1ΔfbaΔtal double mutant was generated and confirmed to be unable to use erythritol. This mutant exhibited a reduced intracellular fitness both in BeWo trophoblasts and THP-1 macrophages. In the murine model, Rev1ΔfbaΔtal provided comparable protection to the Rev1 reference vaccine while inducing fewer adverse reproductive events in pregnant animals. Altogether, these results postulate the Rev1ΔfbaΔtal mutant as a reproductively safer Rev1-derived vaccine candidate to be studied in the natural host. Full article

Brucellosis is an important bacterial disease of livestock and the most common zoonotic disease. The current vaccines are effective but unsafe, as they result in animal abortions and are pathogenic to humans. Virus-like particles are being investigated as molecular scaffolds for foreign antigen presentation to the immune system. Here, we sought to develop a new-generation vaccine by presenting selected Brucella melitensis T cell epitopes on the surface of Orbivirus core-like particles (CLPs) and transiently expressing these chimeric particles in Nicotiana benthamiana plants. We successfully demonstrated the assembly of five chimeric CLPs in N. benthamiana plants, with each CLP presenting a different T cell epitope. The safety and protective efficacy of three of the highest-yielding CLPs was investigated in a mouse model of brucellosis. All three plant-expressed chimeric CLPs were safe when inoculated into BALB/c mice at specific antigen doses. However, only one chimeric CLP induced protection against the virulent Brucella strain challenge equivalent to the protection induced by the commercial Rev1 vaccine. Here, we have successfully shown the assembly, safety and protective efficacy of plant-expressed chimeric CLPs presenting B. melitensis T cell epitopes. This is the first step in the development of a safe and efficacious subunit vaccine against brucellosis. Full article

Background: Brucella melitensis is recognized as one of the predominant zoonotic pathogens globally. Live-attenuated vaccine Rev 1 is currently the most effective vaccine for controlling B. melitensis in small ruminants. While Brucella inactivated, nanoparticle, and subunit vaccines are less effective and require multiple doses, live-attenuated vaccines are less expensive and more efficacious. Several drawbacks are associated with the administration of current attenuated B. melitensis vaccines, including interference with serological diagnostic tests, inducing abortion in pregnant animals, shedding in milk, and zoonotic infections in humans. In this systematic review, we summarize the current literature (1970–2022) on B. melitensis vaccines and review their advantages and disadvantages in order to support the rationale for a need for new or improved small ruminant brucellosis vaccines. Methods: A systematic search was carried out in Web of Science, CAB Abstracts, and PubMed. The original articles describing the B. melitensis vaccines were included. Review articles, articles not published in English, articles that did not offer full text, editorials, correspondences, case reports, case series, diagnostic tests, duplicate publications, and other Brucella vaccines (e.g., B. abortus and B. suis) were excluded. Results: Out of 3700 studies, we identified 18 articles that evaluated B. meltensis vaccines, including recombinant B. melitensis strains (16MΔhfq, 16MΔTcfSR, M5-90ΔmanB, LVM31, M5-90ΔvjbR, 16MΔmucR, ΔznuA, M5-90Δpgm, M5-90ΔwboA), live B. melitensis strain (Rev 1), nanoparticle vaccines (B. melitensis 16M, B. melitensis OMP 31, FliC protein—Mannosylated Chitosan Nanoparticles (FliC and FliC-MCN), B. melitensis and B. abortus combined, and B. melitensis 16M nanoparticles combined with oligopolysaccharide), subunit vaccines (outer membrane vesicles or outer membrane proteins), and a DNA vaccine based on B. melitensis outer membrane proteins (Omp25 and Omp31). The results from these studies revealed that these vaccines can induce humoral and cellular responses and reduce macrophage survival. However, most of these vaccines were evaluated only in murine models, which may not accurately reflect how they work in natural hosts. Conclusions: The high prevalence of B. melitensis in humans and animals remains an issue in many parts of the world. Human brucellosis can be prevented by controlling brucellosis in livestock using vaccination and test-and-removal strategies. Prospective vaccines have limitations, including interference with serodiagnostics after vaccination, virulence in humans and animals, the requirement of booster vaccinations, and insufficient efficacy in preventing infection or abortion. Moreover, most of these vaccines have been assessed in mice models, which have failed to predict immunogenicity or efficacy in natural hosts. Because of these limitations and the re-emergence of B. melitensis worldwide with a high incidence of human infection, our review suggests a need for additional research into the molecular pathology and immunological properties of B. melitensis infection and the identification of protective epitopes or genes that would allow for the development of improved vaccines for small ruminants. Full article

Brucellosis remains an important zoonotic disease in several parts of the world; in Greece, although it is declining, it is still endemic, affecting both the financial and public health sectors. The current study was undertaken to investigate the presence and distribution of virulence-associated genes among Brucella spp. clinical strains isolated during 2001–2022. Species identification was performed using conventional methodology and Bruce-ladder PCR. The presence of the virulence genes mviN, manA, wbkA, perA, omp19, ure, cbg and virB was investigated using PCR. During the study period, a total of 334 Brucella isolates were identified, of which 328 (98.2%) were detected from positive blood cultures; 315 (94.3%) of the isolates were identified as B. melitensis, whilst the remaining 16 (4.8%) and 3 (0.9%) were identified as B. abortus and B. suis, respectively. Notably, two of the B. melitensis were assigned to the REV-1 vaccine strain type. The presence of the omp19, manA, mviN and perA genes was confirmed in all 315 B. melitensis isolates, while ure, wbkA, cbg and virB genes were detected in all but 9, 2, 1 and 1 of the isolates, respectively. All eight virulence genes were amplified in all B. abortus and B. suis isolates. The detection rate of virulence genes did not differ significantly among species. In conclusion, brucellosis is still considered a prevailing zoonotic disease in Greece, with the majority of the isolates identified as B. melitensis. The eight pathogenicity-associated genes were present in almost all Brucella isolates, although the ure gene was absent from a limited number of B. melitensis isolates. Full article

Serological diagnosis provides a robust and effective approach to monitoring and controlling small ruminant brucellosis. Brucella melitensis Rev. 1 is a live vaccine strain used in prophylactic vaccination against small ruminant brucellosis. Because the vaccine strain shares identical serological antigens with the corresponding field strains, differentiating infected from vaccinated animals (DIVA) serological responses hamper surveillance campaigns and interventions that involve vaccination. We have developed a serum PCR-based approach in which we amplify and sequence Brucella omp2a as a DIVA solution and tRNA (uracil-5-)-methyltransferase as a species marker in the serum samples to determine the etiological agent involved in brucellosis field cases. Using this method, we identified the involvement of both the Rev. 1 vaccine strain and a field strain in an outbreak of brucellosis in a flock. This method represents a novel approach in studying the etiology of brucellosis using serum samples as a source of the pathogen’s DNA. Full article

Abortion and reproductive failures induced by Brucella are the main symptoms of animal brucellosis. Laboratory animal models are essential tools of research to study the Brucella pathogenesis before experimentation in natural hosts. To extend the existing knowledge, we studied B. melitensis 16M (virulent) and Rev1 (attenuated) as well as B. suis bv2 infections in pregnant mice. Here, we report new information about kinetics of infection (in spleens, blood, placentas, vaginal shedding, and foetuses), serum cytokine profiles, and histopathological features in placentas and the litter throughout mice pregnancy. Both B. melitensis strains showed a marked placental tropism and reduced viability of pups (mainly in 16M infections), which was preceded by an intense Th1-immune response during placental development. In contrast, B. suis bv2 displayed lower placental tropism, mild proinflammatory immune response, and scarce bacterial transmission to the litter, thus allowing foetal viability. Overall, our studies revealed three different smooth Brucella patterns of placental and foetal pathogenesis in mice, providing a useful animal model for experimental brucellosis. Full article

Vaccination against brucellosis using live attenuated strains is the primary approach in protecting livestock against the disease through a strong cellular immune response. Attenuated vaccine strains also induce serum anti-Brucella antibodies, mostly against Brucella O-polysaccharide, but their role in protection against the disease remains unclear. In this study, we show that Brucella OPS serum antibodies after vaccination or natural infection could kill Brucella in vitro as shown by the serum bactericidal activity (SBA) assay. We used serum samples of Rev. 1 vaccinated sheep that were negative or positive for Brucella OPS antibodies by either one of complement fixation test (CFT), microplate agglutination test (MAT) and ELISA, or sera of naturally infected sheep positive by CFT. We found a significant increase in the killing ability of sera 30 days after intraocular vaccination with Rev. 1 as compared with pre-vaccination. SBA was significantly higher in sera containing Brucella OPS IgG antibodies in comparison with sera lacking such antibodies (p < 0.001 against 16M & Rev. 1 strains). All 10 sera of convalescent sheep demonstrated significant killing ability against the 16M B. melitensis field strain. Specific OPS antibodies participate in the in vitro complement mediated Brucella killing suggesting a potential role in protection against the disease through this mechanism and relevance of developing OPS-based Brucella vaccines. Full article

Brucella sp. are the causative agents of brucellosis. One of the main characteristics of the Brucella genus concerns its very high genetic homogeneity. To date, classical bacteriology typing is still considered as the gold standard assay for direct diagnosis of Brucella. Molecular approaches are routinely used for the identification of Brucella at the genus level. However, genotyping is more complex, and to date, no method exists to quickly assign a strain into species and biovar levels, and new approaches are required. Next generation sequencing (NGS) opened a new era into the diagnosis of bacterial diseases. In this study, we designed a high-resolution melting (HRM) method for the rapid screening of DNA and direct assignment into one of the 12 species of the Brucella genus. This method is based on 17 relevant single nucleotide polymorphisms (SNPs), identified and selected from a whole genome SNP (wgSNP) analysis based on 988 genomes (complete and drafts). These markers were tested against the collection of the European Reference Laboratory (EU-RL) for brucellosis (1440 DNAs extracted from Brucella strains). The results confirmed the reliability of the panel of 17 SNP markers, allowing the differentiation of each species of Brucella together with biovars 1, 2, and 3 of B. suis and vaccine strain Rev1 (B. melitensis) within 3 h, which is a considerable gain of time for brucellosis diagnosis. Therefore, this genotyping tool provides a new and quick alternative for Brucella identification based on SNPs with the HRM-PCR assay. Full article

Brucella species are facultative intracellular bacteria that cause brucellosis, a zoonotic world-wide disease. The live attenuated B. melitensis Rev.1 vaccine strain is widely used for the control of brucellosis in the small ruminant population. However, Rev.1 induces antibodies against the O-polysaccharide (O-PS) of the smooth lipopolysaccharide thus, it is difficult to differentiate between infected and vaccinated animals. Hence, rough Brucella strains lacking the O-PS have been introduced. In the current study, we conducted a comprehensive comparative analysis of the genome sequence of two natural Rev.1 rough strains, isolated from sheep, against that of 24 Rev.1 smooth strains and the virulent reference strain B. melitensis 16M. We identified and characterized eight vital mutations within highly important genes associated with Brucella lipopolysaccharide (LPS) biosynthesis and virulence, which may explain the mechanisms underlying the formation of the Rev.1 rough phenotype and may be used to determine the mechanism underlying virulence attenuation. Further complementation studies aimed to estimate the specific role of these mutations in affecting Brucella morphology and virulence will serve as a basis for the design of new attenuated vaccines for animal immunization against brucellosis. Full article

Brucellosis is a common zoonotic disease of major concern in humans of Kuwait, and B. melitensis causes most human cases. The disease is endemic in small ruminants, cattle, and camels for decades, causing substantial economic losses in livestock production. However, a nationwide large-scale investigation of brucellosis in the small ruminant population has not been done in the past two decades. A serosurvey of sheep brucellosis in the five districts of Kuwait with most animal production farms was done between 2016 and 2019. In total, 67,054 serum samples from 233 sheep herds were collected and tested. Additionally, milk and tissue samples were collected from 46 seropositive cases for bacteriology. Thirty persons from seven seropositive farms were tested by serology. The incidence of seropositive cases was 7% in districts devoid of vaccination, while it was 4.7% in farms with history of vaccination. The serosurvey revealed that 89% of non-vaccinated herds (n = 181) were seropositive by Rose Bengal test (RBT), buffered acidified plate antigen test (BAPAT), and complement fixation test (CFT). Prevalence of 100% was reported for non-vaccinated sheep herds from Al-Wafrah and Al-Jahra districts, followed by those from Al-Salmi (88.24%), Al-Abdali (86.7%) and Kabd (75.6%). Implementation of vaccination with B. melitensis Rev.1 vaccine and test-and-slaughters in 20 herds reduced the seroprevalence to 33.3% and 25% in herds from Al-Jahra and AL-Wafrah, respectively. B. melitensis was isolated from 20 samples (43.5%). More than half of the examined animal owners (56.6%) tested positive for Brucella using RBT, BAPAT and CFT. The high numbers of infected herds and high prevalence in herdsmen are alarming. Thus, control measures have to be ensured immediately. The epidemiological situation in Kuwait is similar to those of the neighboring countries and the combined action of these states is needed. The understanding of the economic and public health impact of brucellosis in Kuwait needs to grow. Full article

The intracellular pathogenic bacteria belonging to the genus Brucella must cope with acidic stress as they penetrate the host via the gastrointestinal route, and again during the initial stages of intracellular infection. A transcription-level regulation has been proposed to explain this but the specific molecular mechanisms are yet to be determined. We recently reported a comparative transcriptomic analysis of the attenuated vaccine Brucella melitensis strain Rev.1 against the virulent strain 16M in cultures grown under either neutral or acidic conditions. Here, we re-analyze the RNA-seq data of 16M from our previous study and compare it to published transcriptomic data of this strain from both an in cellulo and an in vivo model. We identify 588 genes that are exclusively differentially expressed in 16M grown under acidic versus neutral pH conditions, including 286 upregulated genes and 302 downregulated genes that are not differentially expressed in either the in cellulo or the in vivo model. Of these, we highlight 13 key genes that are known to be associated with a bacterial response to acidic stress and, in our study, were highly upregulated under acidic conditions. These genes provide new molecular insights into the mechanisms underlying the acid-resistance of Brucella within its host. Full article