Evaluation of the immunization of camels with Brucella abortus vaccine (RB51) in Egypt.

Background: Brucellosis is a highly contagious zoonotic disease caused by an intracellular facultative microorganism termed Brucella spp. Control of brucellosis depends on test and slaughter policy as well as vaccination programs. Aim: Estimation of the cell-mediated immunity (CMI) [total leukocytic count (TLC), phagocytic activity, phagocytic index, interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α)] in camels after vaccination with RB51 using real-time polymerase chain reaction (PCR). Methods: A total of eight camels were grouped into two groups as follows: group (A): vaccinated with RB51 vaccine [1 dose/2 ml S/C (3 × 1010 CFU)] and group (B): control group. IL-6 and TNF-α were used for estimation of the CMI using real-time PCR on serum samples that were collected at 0, 7, 14, 21, 28, and 60 days after vaccination from each group. In addition, TLC, phagocytic activity, and phagocytic index were evaluated on heparinized blood samples at 0 and 60 days post-vaccination. Results: RB51 vaccine provides a protective immune response which progressively increases from the first week to 60 days after vaccination. Moreover, the levels of TNF-α and IL-6 differed between camels in the vaccinated group. Conclusion: Vaccination of camels with RB51 vaccine (with dose 3 × 1010 CFU) could induce good protective immune responses and this immunological response will be a good indication for a safe field vaccine that can be used for the control of camel brucellosis.

Brucella abortus triggers the differential expression of immunomodulatory lncRNAs in infected murine macrophages.

Introduction: The lncRNAs (long non-coding RNAs) are the most diverse group of non-coding RNAs and are involved in most biological processes including the immune response. While some of them have been recognized for their influence on the regulation of inflammatory activity, little is known in the context of infection by Brucella abortus, a pathogen that presents significant challenges due to its ability to manipulate and evade the host immune system. This study focuses on characterize the expression profile of LincRNA-cox2, Lethe, lincRNA-EPS, Malat1 and Gas5 during infection of macrophages by B. abortus. Methods: Using public raw RNA-seq datasets we constructed for a lncRNA expression profile in macrophages Brucella-infected. In addition, from public RNA-seq raw datasets of RAW264.7 cells infected with B. abortus we constructed a transcriptomic profile of lncRNAs in order to know the expression of the five immunomodulating lncRNAs studied here at 8 and 24 h post-infection. Finally, we performed in vitro infection assays in RAW264.7 cells and peritoneal macrophages to detect by qPCR changes in the expression of these lncRNAs at first 12 hours post infection, a key stage in the infection cycle where Brucella modulates the immune response to survive. Results: Our results demonstrate that infection of macrophages with Brucella abortus, induces significant changes in the expression of LincRNA-Cox2, Lethe, LincRNA-EPS, Gas5, and Malat1. Discussion: The change in the expression profile of these immunomodulatory lncRNAs in response to infection, suggest a potential involvement in the immune evasion strategy employed by Brucella to facilitate its intracellular survival.

Gated nanoprobe utilizing metal-organic frameworks for identifying and distinguishing between the wild strains and the vaccine strains of brucella.

In the assay for Brucella, the identification and differentiation of wild strains and vaccine strains present a significant challenge. Currently, there aren't any commercially available product to address this issue. In this study, we have developed a novel gated nanoprobe by utilizing Metal-Organic Frameworks (MOFs) as a scaffold and hairpin DNA as a "gating switch". Specifically, Probe 1 with hairpin structure (P1h) targets a gene that is present in both wild strains Y3 (B. melitensis biovar 3) and vaccine strains A19 (Brucella abortus strains A19). We successfully applied this probe to screen positive samples of Brucella without any cross-reactivity with other substances. Additionally, we identified another specific gene exclusively found in wild strains, which serves as Probe 2 with hairpin structure (P2h) to confirm the strain type. Simultaneous detachment of both P1h and P2h from the MOFs leads to the release of Rhodamine 6G (Rho 6G) and Fluorescein (Flu), specifically indicating the presence of wild strains. If only P1h detaches and the Flu signal is detected, it suggests the presence of vaccine strains. Importantly, this method offers high accuracy, with a detection rate of 90% and a recovery rate of 94.71% to 107.65%, while avoiding cross-reactions with MO and TB. This one-step experiment provides reliable identification and differentiation of Y3 and A19, addressing concerns related to long periodicity, interference from individual variations, and the complex design of primers in existing laboratory methods. Furthermore, our approach successfully detects target 1 (T1) and target 2 (T2) at concentrations ranging from 10-6 M to 10-9 M, with a detection limit of 6.7 × 10-10 M and 6.4 × 10-10 M, respectively. Importantly, our strategy is cost-effective (around $1) and offers higher detection efficiency compared to traditional laboratory methods.

Guanylate-binding protein-5 is involved in inflammasome activation by bacterial DNA but only the cooperation of multiple GBPs accounts for control of Brucella abortus infection.

Introduction: Guanylate-binding proteins (GBPs) are produced in response to pro-inflammatory signals, mainly interferons. The most studied cluster of GBPs in mice is on chromosome 3. It comprises the genes for GBP1-to-3, GBP5 and GBP7. In humans, all GBPs are present in a single cluster on chromosome 1. Brucella abortus is a Gram-negative bacterium known to cause brucellosis, a debilitating disease that affects both humans and animals. Our group demonstrated previously that GBPs present on murine chromosome 3 (GBPchr3) is important to disrupt Brucella-containing vacuole and GBP5 itself is important to Brucella intracellular LPS recognition. In this work, we investigated further the role of GBPs during B. abortus infection. Methods and results: We observed that all GBPs from murine chromosome 3 are significantly upregulated in response to B. abortus infection in mouse bone marrow-derived macrophages. Of note, GBP5 presents the highest expression level in all time points evaluated. However, only GBPchr3-/- cells presented increased bacterial burden compared to wild-type macrophages. Brucella DNA is an important Pathogen-Associated Molecular Pattern that could be available for inflammasome activation after BCV disruption mediated by GBPs. In this regard, we observed reduced IL-1ß production in the absence of GBP2 or GBP5, as well as in GBPchr3-/- murine macrophages. Similar result was showed by THP-1 macrophages with downregulation of GBP2 and GBP5 mediated by siRNA. Furthermore, significant reduction on caspase-1 p20 levels, LDH release and Gasdermin-D conversion into its mature form (p30 N-terminal subunit) was observed only in GBPchr3-/- macrophages. In an in vivo perspective, we found that GBPchr3-/- mice had increased B. abortus burden and higher number of granulomas per area of liver tissue, indicating increased disease severity. Discussion/conclusion: Altogether, these results demonstrate that although GBP5 presents a high expression pattern and is involved in inflammasome activation by bacterial DNA in macrophages, the cooperation of multiple GBPs from murine chromosome 3 is necessary for full control of Brucella abortus infection.

Effects of the Immunocontraceptive Gonacon on Pregnancy in Brucella-Seropositive American bison (Bison bison).

The purpose of this study was to determine if the number of pregnancies in naturally infected Brucella abortus-positive bison (Bison bison) cows would be reduced over a period of 5 yr after one treatment with 3000 µg gonadotropin-releasing hormone immunocontraceptive (GonaCon) compared to a similar group of naturally infected B. abortus-positive bison cows not treated with GonaCon. In each of the 5 yr, GonaCon-treated cows produced fewer offspring in relation to number of cows than the nontreated cows. Fisher's Exact test comparing offspring produced during the first reproductive season showed a significant difference between the two groups (P=0.0028). Differences in number of calves produced in GonaCon-treated and control groups were also noted in remaining years, but statistics were not applied because of data constraints. These data indicate that one treatment with GonaCon in brucellosis-seropositive female bison reduced pregnancies over five reproductive years. Thus, immunocontraception could potentially be used to manage brucellosis in affected herds.

Effects of Pregnancy Prevention on Brucella abortus Shedding in American bison (Bison bison).

Products of parturition are the predominant source of Brucella abortus for transmission in bison (Bison bison). Our objective was to assess whether preventing pregnancy in Brucella-seropositive bison reduced B. abortus shedding. Brucella-seropositive and -seronegative bison from Yellowstone National Park, Wyoming, USA were used in a replicated experiment. Each of two replicates (rep1, rep2) included a group of seropositive females treated with a single dose of gonadotropin-releasing hormone-based immunocontraceptive (Treatment rep1, n=15; Treatment rep2, n=20) and an untreated group (Control rep1, n=14; Control rep2, n=16) housed separately. Seronegative sentinel females were placed in each group to monitor horizontal transmission. Seronegative males were co-mingled for breeding each year. Pregnant females were removed from treatment groups in the first year, but not thereafter. Each January-June we monitored for B. abortus shedding events-any parturition associated with culture-positive fluids or tissues. We analyzed probability of shedding events using a negative binomial generalized linear mixed model fit by maximum likelihood using Laplace approximation. Over 5 yr, we observed zero shedding events in Treatment rep1 vs. 12 in Control rep1. All five Control rep1 sentinels but zero (0/5) Treatment rep1 sentinels seroconverted. In the second replicate, Treatment rep2 had two shedding events over 3 yr and Control rep2 had five events over 2 yr. Sentinels in both Control rep2 (3/6) and Treatment rep2 (5/6) seroconverted by trial endpoint. Treatment rep1 showed a reduced shedding probability relative to Control rep1, Treatment rep2, and Control rep2 (log odds value -25.36 vs. -1.71, -1.39, and -0.23, respectively). Fixed effect predictor covariates, year and age, had no explanatory value. These data suggest that successful contraception of brucellosis-seropositive female bison prevents shedding of B. abortus by individual animals. However, contraceptive treatment may or may not sufficiently reduce disease transmission to reduce brucellosis prevalence in an affected herd.

Contrasting roles for IgM and B-cell MHCII expression in Brucella abortus S19 vaccine-mediated efficacy against B. melitensis infection.

Brucellosis, caused by the bacterium Brucella, poses a significant global threat to both animal and human health. Although commercial live Brucella vaccines including S19, RB51, and Rev1 are available for animals, their unsuitability for human use and incomplete efficacy in animals necessitate the further study of vaccine-mediated immunity to Brucella. In this study, we employed in vivo B-cell depletion, as well as immunodeficient and transgenic mouse models, to comprehensively investigate the roles of B cells, antigen uptake and presentation, antibody production, and class switching in the context of S19-mediated immunity against brucellosis. We found that antibody production, and in particular secretory IgM plays a protective role in S19-mediated immunity against virulent Brucella melitensis early after the challenge in a manner associated with complement activation. While T follicular helper cell deficiency dampened IgG production and vaccine efficacy at later stages of the challenge, this effect appeared to be independent of antibody production and rather was associated with altered T-cell function. By contrast, B-cell MHCII expression negatively impacted vaccine efficacy at later timepoints after the challenge. In addition, B-cell depletion after vaccination, but before the challenge, enhanced S19-mediated protection against brucellosis, suggesting a deleterious role of B cells during the challenge phase. Collectively, our findings indicate antibody production is protective, while B-cell MHCII expression is deleterious, to live vaccine-mediated immunity against brucellosis. IMPORTANCE: Brucella is a neglected zoonotic pathogen with a worldwide distribution. Our study delves into B-cell effector functions in live vaccine-mediated immunity against brucellosis. Notably, we found antibody production, particularly secretory IgM, confers protection against virulent Brucella melitensis in vaccinated mice, which was associated with complement activation. By contrast, B-cell MHCII expression negatively impacted vaccine efficacy. In addition, B-cell depletion after vaccination, but before the B. melitensis challenge, enhanced protection against infection, suggesting a detrimental B-cell role during the challenge phase. Interestingly, deficiency of T follicular helper cells, which are crucial for aiding germinal center B cells, dampened vaccine efficacy at later stages of challenge independent of antibody production. This study underscores contrasting and phase-dependent roles of B-cell effector functions in vaccine-mediated immunity against Brucella.

Comparative genomics of Brucella abortus and Brucella melitensis unravels the gene sharing, virulence factors and SNP diversity among the standard, vaccine and field strains / La genómica comparada de Brucella abortus y Brucella melitensis revela el intercambio de genes, los factores de virulencia y la diversidad de SNP entre las cepas estándar, vacunales y de campo

Brucella abortus and Brucella melitensis are the primary etiological agents of brucellosis in large and small ruminants, respectively. There are limited comparative genomic studies involving Brucella strains that explore the relatedness among both species. In this study, we involved strains (n=44) representing standard, vaccine and Indian field origin for pangenome, single nucleotide polymorphism (SNP) and phylogenetic analysis. Both species shared a common gene pool representing 2884 genes out of a total 3244 genes. SNP-based phylogenetic analysis indicated higher SNP diversity among B. melitensis (3824) strains in comparison to B. abortus (540) strains, and a clear demarcation was identified between standard/vaccine and field strains. The analysis for virulence genes revealed that virB3, virB7, ricA, virB5, ipx5, wbkC, wbkB, and acpXL genes were highly conserved in most of the Brucella strains. Interestingly, virB10 gene was found to have high variability among the B. abortus strains. The cgMLST analysis revealed distinct sequence types for the standard/vaccine and field strains. B. abortus strains from north-eastern India fall within similar sequence type differing from other strains. In conclusion, the analysis revealed a highly shared core genome among two Brucella species. SNP analysis revealed B. melitensis strains exhibit high diversity as compared to B. abortus strains. Strains with absence or high polymorphism of virulence genes can be exploited for the development of novel vaccine candidates effective against both B. abortus and B. melitensis.(AU)

Structure-function analysis of the cyclic ß-1,2-glucan synthase from Agrobacterium tumefaciens.

The synthesis of complex sugars is a key aspect of microbial biology. Cyclic ß-1,2-glucan (CßG) is a circular polysaccharide critical for host interactions of many bacteria, including major pathogens of humans (Brucella) and plants (Agrobacterium). CßG is produced by the cyclic glucan synthase (Cgs), a multi-domain membrane protein. So far, its structure as well as the mechanism underlining the synthesis have not been clarified. Here we use cryo-electron microscopy (cryo-EM) and functional approaches to study Cgs from A. tumefaciens. We determine the structure of this complex protein machinery and clarify key aspects of CßG synthesis, revealing a distinct mechanism that uses a tyrosine-linked oligosaccharide intermediate in cycles of polymerization and processing of the glucan chain. Our research opens possibilities for combating pathogens that rely on polysaccharide virulence factors and may lead to synthetic biology approaches for producing complex cyclic sugars.

A novel Bayesian Latent Class Model (BLCM) evaluates multiple continuous and binary tests: A case study for Brucella abortus in dairy cattle.

Bovine brucellosis, primarily caused by Brucella abortus, severely affects both animal health and human well-being. Accurate diagnosis is crucial for designing informed control and prevention measures. Lacking a gold standard test makes it challenging to determine optimal cut-off values and evaluate the diagnostic performance of tests. In this study, we developed a novel Bayesian Latent Class Model that integrates both binary and continuous testing outcomes, incorporating additional fixed (parity) and random (farm) effects, to calibrate optimal cut-off values by maximizing Youden Index. We tested 651 serum samples collected from six dairy farms in two regions of Henan Province, China with four serological tests: Rose Bengal Test, Serum Agglutination Test, Fluorescence Polarization Assay, and Competitive Enzyme-Linked Immunosorbent Assay. Our analysis revealed that the optimal cut-off values for FPA and C-ELISA were 94.2 mP and 0.403 PI, respectively. Sensitivity estimates for the four tests ranged from 69.7% to 89.9%, while specificity estimates varied between 97.1% and 99.6%. The true prevalences in the two study regions in Henan province were 4.7% and 30.3%. Parity-specific odds ratios for positive serological status ranged from 1.2 to 2.2 for different parity groups compared to primiparous cows. This approach provides a robust framework for validating diagnostic tests for both continuous and discrete tests in the absence of a gold standard test. Our findings can enhance our ability to design targeted disease detection strategies and implement effective control measures for brucellosis in Chinese dairy farms.

In vivo biological validation of in silico analysis: A novel approach for predicting the effects of TLR4 exon 3 polymorphisms on brucellosis.

The role of the Toll-like receptor 4 (TLR4) is of recognising intracellular and extracellular pathogens and of activating the immune response. This process can be compromised by single nucleotide polymorphisms (SNPs) which might affect the activity of several TLRs. The aim of this study is of ascertaining whether SNPs in the TLR4 of Bubalus bubalis infected by Brucella abortus, compromise the protein functionality. For this purpose, a computational analysis was performed. Next, computational predictions were confirmed by performing genotyping analysis. Finally, NMR-based metabolomics analysis was performed to identify potential biomarkers for brucellosis. The results indicate two SNPs (c. 672 A > C and c. 902 G > C) as risk factor for brucellosis in Bubalus bubalis, and three metabolites (lactate, 3-hydroxybutyrate and acetate) as biological markers for predicting the risk of developing the disease. These metabolites, together with TLR4 structural modifications in the MD2 interaction domain, are a clear signature of the immune system alteration during diverse Gram-negative bacterial infections. This suggests the possibility to extend this study to other pathogens, including Mycobacterium tuberculosis. In conclusion, this study combines multidisciplinary approaches to evaluate the biological and structural effects of SNPs on protein function.

Prevalence of Brucella melitensis and Brucella abortus aminoglycoside-resistant isolates: a systematic review and meta-analysis.

INTRODUCTION: Aminoglycosides are vital antibiotics for treating Brucella infections, because they interfere with bacterial protein production and are often combined with other antibiotics. They are cost-effective, have fewer side effects, and can penetrate biofilms. The prevalence of brucellosis has increased in recent years, increasing the need for effective treatments. In addition, the emergence of multidrug-resistant Brucella strains has highlighted the need for an updated and comprehensive understanding of aminoglycoside resistance. This systematic review aimed to provide a comprehensive overview of the global prevalence of aminoglycoside resistance in B. melitensis and B. abortus. METHODS: A systematic search of online databases was conducted and eligible studies met certain criteria and were published in English. Quality assessment was performed using the JBI Checklist. A random-effects model was fitted to the data, and meta-regression, subgroup, and outlier/influential analyses were performed. The analysis was performed using R and the metafor package. RESULTS: The results of this systematic review and meta-analysis suggested that the average prevalence rates of streptomycin, gentamicin, and amikacin resistance were 0.027 (95% confidence interval [CI], 0.015-0.049), 0.023 (95% CI, 0.017-0.032), and 0.008 (95% CI, 0.002-0.039), respectively. The prevalence of streptomycin resistance was higher in the unidentified Brucella group than in the B. abortus and B. melitensis groups (0.234, 0.046, and 0.017, respectively; p < 0.02). The prevalence of gentamicin resistance increased over time (r = 0.064; 95% CI, 0.018 to 0.111; p = 0.007). The prevalence of resistance did not correlate with the quality score for any antibiotic. Funnel plots showed a potential asymmetry for streptomycin and gentamicin. These results suggest a low prevalence of antibiotic resistance in the studied populations. CONCLUSION: The prevalence of aminoglycoside resistance in B. melitensis and B. abortus was low. However, gentamicin resistance has increased in recent years. This review provides a comprehensive and updated understanding of aminoglycoside resistance in B. melitensis and B. abortus.

Comparative genomics of Brucella abortus and Brucella melitensis unravels the gene sharing, virulence factors and SNP diversity among the standard, vaccine and field strains.

Brucella abortus and Brucella melitensis are the primary etiological agents of brucellosis in large and small ruminants, respectively. There are limited comparative genomic studies involving Brucella strains that explore the relatedness among both species. In this study, we involved strains (n=44) representing standard, vaccine and Indian field origin for pangenome, single nucleotide polymorphism (SNP) and phylogenetic analysis. Both species shared a common gene pool representing 2884 genes out of a total 3244 genes. SNP-based phylogenetic analysis indicated higher SNP diversity among B. melitensis (3824) strains in comparison to B. abortus (540) strains, and a clear demarcation was identified between standard/vaccine and field strains. The analysis for virulence genes revealed that virB3, virB7, ricA, virB5, ipx5, wbkC, wbkB, and acpXL genes were highly conserved in most of the Brucella strains. Interestingly, virB10 gene was found to have high variability among the B. abortus strains. The cgMLST analysis revealed distinct sequence types for the standard/vaccine and field strains. B. abortus strains from north-eastern India fall within similar sequence type differing from other strains. In conclusion, the analysis revealed a highly shared core genome among two Brucella species. SNP analysis revealed B. melitensis strains exhibit high diversity as compared to B. abortus strains. Strains with absence or high polymorphism of virulence genes can be exploited for the development of novel vaccine candidates effective against both B. abortus and B. melitensis.

Serological and Molecular Survey of Brucella Species in Owners and Their Dogs Living on Island and Mainland Seashore Areas of Brazil.

Background: Although Brucella abortus, Brucella suis, and Brucella canis may infect humans and dogs worldwide, no study to date has assessed and compared owners and their dogs between island and mainland seashore areas. Materials and Methods: Accordingly, the study herein has applied serological tests, including Microplate Agglutination Test with 2-Mercaptoethanol, immunochromatographic assay, and Rose Bengal Test, and a Brucella genus-specific PCR assay to 195 owners and their 148 dogs living on 1 mainland seashore area and three nearby oceanic islands of southern Brazil. Results: No seropositivity to B. abortus and B. suis was detected in owner or dog sera. Anti-B. canis seropositivity was observed in 3/148 (2.0%) dogs, but no owner sample was seropositive to B. canis. In addition, all blood samples from both owners and dogs were negative on Brucella genus-specific PCR assay. Conclusions: The seropositive dogs were not related and lived on the seashore mainland area of Guaraqueçaba city. The absence of seropositivity on the islands and the low seropositivity on the seashore mainland could be attributed to geographic isolation, and suggest the low impact of the disease in the region. Despite being a zoonotic disease, brucellosis by B. canis is not included in the National Program for Control and Eradication of Brucellosis, and its diagnosis and notification are not mandatory. The presence of seropositive dogs highlights the risk to human health and the importance of epidemiological surveillance actions in the region, as well as the need for the implantation of preventive measures to avoid the transmission of the pathogen.

Reproductive Fate of Brucellosis-Seropositive Elk (Cervus canadensis): Implications for Disease Transmission Risk.

Brucellosis is a disease caused by the bacterium Brucella abortus that infects elk (Cervus canadensis) and cattle (Bos taurus). There is the potential for transmission from wildlife to livestock through contact with infected material shed during abortions or live births. To understand the impact of exposure on pregnancy rates we captured 30-100 elk per year from 2011 through 2020, testing their blood for serologic exposure to B. abortus. Predicted pregnancy rates for seropositive animals were 9.6% lower in prime-age (2.5-15.5 yr; 85%, 95% confidence interval [CI]: 74-91%) and 37.7% lower in old (>15.5 yr; 43%, 95% CI: 19-71%) elk as compared with seronegative animals. To understand the risk of seropositive elk shedding B. abortus bacteria and the effects of exposure on elk reproductive performance, we conducted a 5-yr longitudinal study monitoring 30 seropositive elk. We estimated the annual probability of a seropositive elk having an abortion as 0.06 (95% CI: 0.02-0.15). We detected B. abortus at three abortions and two live births, using a combination of culture and PCR testing. The predicted probability of a pregnant seropositive elk shedding B. abortus during an abortion or live birth was 0.08 (95% CI: 0.04-0.19). To understand what proportion of seropositive elk harbored live B. abortus bacteria in their tissues, we euthanized seropositive elk at the end of 5 yr of monitoring and sampled tissues for B. abortus. Assuming perfect detection, the predicted probability of a seropositive elk having B. abortus in at least one tissue was 0.18 (95% CI: 0.06-0.43). The transmission risk seropositive elk pose is mitigated by decreased pregnancy rates, low probability of abortion events, low probability of shedding at live birth events, and reasonably low probability of B. abortus in tissues.

Effect of selection genotype on immune response to Brucella abortus RB51 in Holstein cattle.

Genetic selection for milk production traits in US Holsteins has affected numerous genes associated with reproduction and immunity. This study compares the transcriptomic response of peripheral blood mononuclear cells to an in vitro Brucella abortus strain RB51 (RB51) bacterial challenge between contemporary Holsteins and Holsteins that have not been selected for milk production traits since the mid-1960s. Total RNA was extracted from peripheral blood mononuclear cells from four contemporary and four unselected lactating, primiparous cows following 24-h incubation with or without stimulation with RB51 bacteria. RNA was sequenced and reads analyzed using tools from galaxy.scinet.usda.gov. A total of 412 differentially expressed genes (false discovery rate p < 0.05, log fold change > |1|) were identified. The upregulated genes (genes with higher expression in contemporary than unselected cattle) were enriched for 19 terms/pathways, including alanine, aspartate, and glutamate metabolism, indicating a cellular stress response. Downregulated genes (genes with higher expression in unselected than contemporary cows) were enriched for 37 terms/pathways, representing diverse immune responses, including natural killer cell-mediated immunity, interferon-γ production, negative regulation of interleukin-10 production, and cytokine receptor activity indicating a broad immune response with an emphasis on immune defense. These results provide evidence that differences exist between the two genotypes in response to in vitro bacterial challenge. This suggests that contemporary cows, genetically selected for milk production, may have reduced immune function, including limitations in response to intracellular bacteria.

Comparative Analysis of Humoral Immune Response and Cognate Antigen Detection in Experimentally Infected Sprague Dawley Rats with Brucella abortus Biotype 1.

Background: This study investigated the IgG-specific humoral immune responses against specific antigen-like whole-cell antigen (WCA), outer membrane protein (OMP), periplasmic protein (PP), and cytoplasmic protein (CP) during the acute and subacute stages of Brucella abortus biotype 1 infection in Sprague Dawley (SD) rats. Materials and Methods: The intraperitoneal method was used to experimentally infect forty-four 6- to 8-week-old SD rats with 1 × 109 colony-forming units (CFUs) of B. abortus biotype 1. Following inoculation, the rat was serially sampled for serum at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90, and 120 days. The IgG-specific immune responses and recognition of immunodominant antigens in WCA, OMP, PP, and CP of B. abortus were assessed by indirect enzyme-linked immunosorbent assay (IELISA) and western blot (WB) assay using infected rat sera. Results: The IgG antibody response was detectable at 3 days after infection. The peak serum IgG antibody titers were recorded against CP and PP at 28 days after infection. The highest serum IgG antibody titers were recorded at 42 days after infection against WCA and 90 days after infection only against OMP. WB assay revealed a wide array of protein bands between molecular weight of 13 and 95 kDa for WCA, 13 and 95 kDa for OMP, 15 and 65 kDa for PP, and 12 and 85 kDa for CP. Proteins bands of 10, 13, 20, 24, 46, and 76 kDa for WCA; 28, 35, 39, 85, and 95 for OMP; 20, 30, 40, 43, 46, and 65 kDa for PP, and 12, 23, 68, and 85 for CP were intensely recognized. Conclusion: Data of this study indicated that WCA, CP, and PP of B. abortus could be useful for diagnosis of acute and subacute brucellosis in SD rat model. OMP of B. abortus could be useful for differential diagnosis of subacute brucellosis.

Is Brucella excreted in cattle faeces? - Evidence from Punjab, India.

Brucellosis is a neglected zoonosis that affects animals and people in much of the underdeveloped world. The disease is endemic in cattle in Punjab, India and controlling it is a public health challenge. Dairy farmers and farm labour commonly handle cattle faeces with bare hands and personal protective equipments are not used. No studies have been conducted about the shedding of Brucella species in faeces of sero positive cattle in the state. This study aimed to isolate and identify the Brucella species from faeces of sero positive cattle in Punjab, India. Faecal samples were collected from 350 Brucella sero positive cattle in Ludhiana district of Punjab, India. Isolation was performed using a pre-enriched Brucella selective broth medium as well as Brucella selective medium agar plates containing horse serum and Brucella selective supplements. Isolates were identified using Gram staining technique and rapid slide agglutination test, and then confirmed by using bcsp31 and 16s rRNA genus specific PCR. Isolates were further identified up to species level by using Bruce-Ladder multiplex PCR. Fourteen Brucella species were isolated, all of which showed coccobacilli on gram staining, positive rapid slide agglutination test and amplification of bcsp31 and 16s rRNA genes. Of the 14 isolates, 11 were identified as Brucella abortus and 3 were identified as Brucella melitensis. The study demonstrates that animal faeces could pose a potential risk for animal and human health and faeces of seropositive cattle must be handled with care.

Prevalence of Brucella melitensis and Brucella abortus Fluoroquinolones Resistant Isolates: A Systematic Review and Meta-Analysis.

Background: Brucellosis impact both animals and humans worldwide. However, using antibiotics for brucellosis remains controversial despite decades of research. Relapse can complicate treatment in this area. Since the mid-1980s, microbiologists, and physicians have studied fluoroquinolones' use for treating human brucellosis. The principal advantages of fluoroquinolones are their intracellular antimicrobial activity, low nephrotoxicity, good pharmacokinetics, and the lack of drug-level monitoring. Fluoroquinolones inhibit disease recurrence. In vitro and clinical data were used to study the prevalence of Brucella melitensis and Brucella abortus fluoroquinolone-resistant isolates. Methods: The PubMed, Scopus, Embase, and Web of Science databases were carefully searched until August 6, 2022, for relevant papers. The number of resistant isolates and sample size were used to estimate the proportion of resistant isolates, fitting a model with random effects, and DerSimonian-Laird estimated heterogeneity. Furthermore, meta-regression and subgroup analyses were used to assess the moderators to identify the sources of heterogeneity. Meta-analysis was performed using R software. Results: Forty-seven studies evaluated fluoroquinolone resistance in Brucella spp. Isolates. Fluoroquinolones have shown high in vitro efficacy against Brucella spp. The resistance rates to ofloxacin, sparfloxacin, fleroxacin, pefloxacin, and lomefloxacin were 2%, 1.6%, and 4.6%, respectively. Conclusion: Clinical in vitro tests demonstrated that fluoroquinolones can eradicate Brucella spp. Owing to first-line medication resistance, recurrence, and toxicity, it is essential to standardize the Brucella antimicrobial susceptibility test method for a more precise screening of resistance status. Fluoroquinolones are less resistant to fluoroquinolone-based treatments in modern clinical practice as alternatives to standard therapy for patients with brucellosis relapse after treatment with another regimen and in patients who have developed toxicity from older agents.