Characterization of innate immune response to Brucella melitensis infection in goats with permissive or restrictive phenotype for Brucella intramacrophagic growth.

Caprine brucellosis is a chronic, world-wide distributed disease which causes reproductive failure in goats and Brucella melitensis, its causative agent, bears a great zoonotic potential. There is evidence suggesting that some cattle and pigs have an innate ability to resist Brucella infection, but this has not yet been investigated in goats. In this study, we compared caprine macrophages that exhibit extreme restriction and permissiveness to B. melitensis' intracellular growth in vitro. Monocyte derived macrophages (MDMs) from 110 female goats were cultured and challenged in vitro with B. melitensis 16 M. After initial screening, 18 donor goats were selected based on their macrophages ability to restrict or allow bacterial intracellular growth and some elements of humoral and cellular immunity were studied in depth. MDMs that were able to restrict the pathogen's intracellular growth showed enhanced bacterial internalization, although there were no differences between groups in the production of reactive oxygen and nitrogen intermediates following 48 h treatment with heat-killed B. melitensis. Moreover, there were no differences between groups in the level of antibodies reacting with keyhole limpet hemocyanin (natural antibodies, NAbs) or with Brucella LPS antigens (cross-reacting antibodies, CrAbs), although a strong positive correlation between individual levels of IgM NAbs and IgM CrAbs was detected. Altogether, these results represent an initial step in understanding innate primary host response to B. melitensis, and deciphering which mechanisms may determine a successful outcome of the infection in goats.

Evaluation of Poly(I:C) and combination of CpG ODN plus Montanide ISA adjuvants to enhance the efficacy of outer membrane vesicles as an acellular vaccine against Brucella melitensis infection in mice.

Brucellosis is the most common zoonotic disease worldwide and still there is no vaccine for human use. The commercial animal vaccines also have major problems that limit their use. Therefore, there is a need for an effective Brucella vaccine which is multivalent and produces a good protective immunity with minimal disadvantages. Due to their heterogeneous composition and diverse functions, OMVs are promising acellular vaccine candidates against brucellosis. In the present study, the potential of Poly(I:C) or CpG ODN 1826+ Montanide ISA 70 VG adjuvant formulations were evaluated to enhance the immunity and protection levels conferred by OMVs against Brucella challenge in mice. The results indicated that both vaccine regimens were able to induce strong Th1-biased responses and confer protective levels significantly higher than REV.1 live vaccine. With regard to the results, it is concluded that OMVs in either adjuvant can be introduced as a new vaccine candidate against B. melitensis infection.

Modelling the immunopathophysiology of Brucella melitensis and its lipopolysaccharide in mice infected via oral route of exposure.

Brucella melitensis is one of the leading zoonotic pathogens with significant economic implications in animal industry worldwide. Lipopolysaccharide, however, remains by far the major virulence with substantial role in diseases pathogenesis. Nonetheless, the effect of B. melitensis and its lipopolysaccharide on immunopathophysiological aspects largely remains an enigma. This study examines the effect of B.melitensis and its lipopolysaccharide on immunopathophysiological parameters following experimental infection using mouse model. Eighty four (n = 84) mice, BALB/c, both sexes with equal gender distribution and 6-8 weeks-old were randomly assigned into three groups. Group 1-2 (n = 72) were orally inoculated with 0.4 mL containing 109 CFU/mL of B. melitensis and its LPS, respectively. Group 3 (n = 12) was challenged orally with phosphate buffered saline and served as a control group. Animals were observed for clinical signs, haematological and histopathological analysis for a period of 24 days post-infection. We hereby report that B.melitensis infected group demonstrated significant clinical signs and histopathological changes than LPS infected group. However, both infected groups showed elevated levels of interleukins (IL-1ß and IL-6) and antibody levels (IgM and IgG) with varying degrees of predominance in LPS infected group than B. melitensis infected group. For hormone analysis, low levels of progesterone, estradiol and testosterone were observed in both B. melitensis and LPS groups throughout the study period. Moreover, in B. melitensis infected group, the organism was re-isolated from the organs and tissues of gastrointestinal, respiratory and reproductive systems thereby confirming the infection and transmission dynamics. This report is the first detailed investigation comparing the infection progression and host responses in relation to the immunopathophysiological aspects in a mouse model after oral inoculation with B. melitensis and its lipopolysaccharide.

Isolation and identification of Brucella melitensis using bacteriological and molecular tools from aborted goats in the Afar region of north-eastern Ethiopia.

BACKGROUND: Infection with Brucella melitensis (B. melitensis) is one of the most important causes of abortion in goats and sheep, and also causes severe systemic disease in exposed humans. In Ethiopia, based on seroepidemiological studies, brucellosis is known to be endemic. However, there is little information on the isolation and molecular detection of Brucella species in small ruminants. Therefore, the present study was conducted in the Amibara district of Afar Region of Ethiopia to isolate and molecularly detect Brucella infection in small ruminants. RESULTS: Out of the total 64 samples cultured, eight samples (five vaginal swabs and three milk) were positive for Brucella species based on colony morphology, growth characteristics, modified acid fast staining and biochemical tests results. Further identification using Brucella- ladder PCR method showed that four of the isolates (three from vaginal swabs and one from milk) from goats amplified fragments of 1071 bp, 794 bp, 587 bp, 450 bp and 152 bp in band size. The molecular result combined with the microbiological and biochemical characteristics of the isolates indicated that the isolates were strains of B. melitensis. CONCLUSION: The finding of this study could suggest economic and zoonotic significance of B. melitensis and warrants for the need for control strategies in livestock and creation of awareness in the pastoral communities on the safe consumption of foods of animal origin and avoidance of physical contact with aborted materials.

The Brucella melitensis M5-90ΔmanB live vaccine candidate is safer than M5-90 and confers protection against wild-type challenge in BALB/c mice.

Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. Although effective, the current Brucella vaccines (strain M5-90 or others) have several drawbacks. The first is their residual virulence for animals and humans and the second is their inability to differentiate natural infection from that caused by vaccination. In the present study, Brucella melitensis M5-90 manB mutant (M5-90ΔmanB) was generated to overcome these drawbacks. M5-90ΔmanB showed significantly reduced survival in macrophages and mice, and induced strong protective immunity in BALB/c mice. It elicited anti-Brucella-specific IgG1 and IgG2a subtype responses and induced the secretion of gamma interferon (IFN-γ) and interleukin-4(IL-4). Results of immune assays showed, M5-90ΔmanB immunization induced the secretion of IFN-γ in goats, and serum samples from goats inoculated with M5-90ΔmanB were negative by Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT). Further, the ManB antigen also allows serological assays differentiate infections caused by wild strains from infections by vaccination. These results show that M5-90ΔmanB is a suitable attenuated vaccine candidate against virulent Brucella melitensis 16 M (16 M) infection.

Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis.

We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment.

[FEATURES OF MASS-SPECTROMETRIC PROTEIN PROFILES OF STRAINS OF BRUCELLOSIS CAUSATIVE AGENT DURING PREPARATION OF CULTURE ON VARIOUS NUTRIENT MEDIA].

AIM: Carry out comparative analysis using time-of-flight mass-spectrometry with matrix laser desorption/ionization (MALDI-TOF MS) of protein profiles of brucellosis causative agents (Brucella melitensis Rev-1 and Brucella abortus 19BA), cultivated in various nutrient media: Albimi agar, brucellagar and erythrit-agar. MATERIALS AND METHODS: Vaccine,strains: Brucella melitensis Rev-1 and Brucella abortus 19BA. Protein profiling in linear mode on Microflex "Bruker Daltonics" MALDI-TOF mass-spectrometer. RESULTS: A number of characteristic features of brucella mass-spectra was detected: in particular, preservation of the total qualitative composition of protein profiles of cultures and significant differences in the intensity of separate peaks depending on the nutrient medium used. CONCLUSION: Based on the analysis of the data obtained, use of Albimi agar as the nutrient medium for preparation of brucella culture samples for mass-spectrometric analysis was shown to be optimal.

The Brucella melitensis M5-90 phosphoglucomutase (PGM) mutant is attenuated and confers protection against wild-type challenge in BALB/c mice.

Brucellae are Gram-negative intracellular bacterial pathogens that infect humans and animals, bringing great economic burdens to developing countries. Live attenuated Brucella vaccines (strain M5-90 or others) are the most efficient means for prevention and control of animal brucellosis. However, these vaccines have several drawbacks, including residual virulence in animals, and difficulties in differentiating natural infection from vaccine immunization, which limit their application. A vaccine that can differentiate infection from immunization will have extensive applications. A Brucella melitensis (B. melitensis) strain M5-90 pgm mutant (M5-90Δpgm) was constructed to overcome these drawbacks. M5-90Δpgm showed significantly reduced survival in embryonic trophoblast cells and in mice, and induced high protective immunity in BALB/c mice. Moreover, M5-90Δpgm elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon (IFN-γ) and interleukin-2 (IL-2). In addition, M5-90Δpgm induced the secretion of IFN-γ in immunized sheep. Serum samples from sheep inoculated with M5-90Δpgm were negative by the Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT). Furthermore, the PGM antigen allowed serological differentiation between infected and vaccinated animals. These results suggest that M5-90Δpgm is an ideal live attenuated vaccine candidate against B. melitensis 16 M and deserves further evaluation for vaccine development.

A multicopper oxidase contributes to the copper tolerance of Brucella melitensis 16M.

Copper is a potent antimicrobial agent. Multiple mechanisms of copper tolerance are utilized by some pathogenic bacteria. BMEII0580, which is significantly similar to the multicopper oxidase from Escherichia coli, was predicted to be the probable blue copper protein YacK precursor in Brucella melitensis 16M, and was designated as Brucella multicopper oxidase (BmcO). A bioinformatics analysis indicated that the typical motifs of multicopper oxidases are present in BmcO. BmcO, the expression of which was up-regulated by copper, could catalyze the oxidation of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), dimethoxyphenol (DMP) and para-phenylenediamine (pPD), which are widely used as substrates for multicopper oxidase. Additionally, BmcO exhibited ferroxidase activity, which indicated that it might play an important role in the Fe(2+) uptake of B. melitensis. Importantly, the mutant strain 16MΔbmcO was more sensitive to copper than the wild-type strain B. melitensis 16M as well as its complementation strain 16MΔbmcO(bmcO). The infection assays of cells showed that similar bacterial numbers of B. melitensis 16M, 16MΔbmcO and 16MΔbmcO(bmcO) strains were recovered from the infected macrophages. This result indicated that BmcO was not essential for B. melitensis intracellular growth. In conclusion, our results confirm that BmcO is a multicopper oxidase and contributes to the copper tolerance of B. melitensis 16M.

In vitro evaluation of six chemical agents on smooth Brucella melitensis strain.

Brucellosis is a zoonosis that disseminated by a variety of ways between animals and humans. The effective disinfection of contaminated environments, soil, feces, and animal bodies plays an irreplaceable role in the prevention and control of brucellosis. To kill Brucella effectively, the bactericidal effects of frequently used disinfectants (including aldehydes, halogens, quaternary ammonium compound, phenolics, and alkalines) and the potential factors that influence disinfection effects were determined in the present study. The results revealed that the minimum bactericidal concentrations (MBCs) of the six disinfectants were all significantly lower than the routinely used concentrations, and all the tested disinfectants were effective against B. melitensis NI. The results of quantitative determination showed that the bactericidal effects of the disinfectants were influenced by their concentration, exposure time, dirty condition and the temperature. Under dirty conditions and a low temperatures, sodium hypochlorite and sodium hydroxide showed better bactericidal effect, while benzalkonium chloride was almost without bactericidal ability. In addition, increasing the disinfectant concentration at low temperatures can improve the bactericidal effect. The present study suggested that Brucella is sensitive to commonly used disinfectants. However, the bactericidal effect is vulnerable to dirty conditions and low temperatures. Thus, it is necessary to test the in vitro sensitivity of disinfectants that are commonly used on farms or the new disinfectant formulations periodically, with the aim of improving the efficacy of animal and human brucellosis prevention programs.

Decreased in vivo virulence and altered gene expression by a Brucella melitensis light-sensing histidine kinase mutant.

Brucella species utilize diverse virulence factors. Previously, Brucella abortus light-sensing histidine kinase was identified as important for cellular infection. Here, we demonstrate that a Brucella melitensis LOV-HK (BM-LOV-HK) mutant strain has strikingly different gene expression than wild type. General stress response genes including the alternative sigma factor rpoE1 and its anti-anti-sigma factor phyR were downregulated, while flagellar, quorum sensing (QS), and type IV secretion system genes were upregulated in the ΔBM-LOV-HK strain vs. wild type. Contextually, expression results agree with other studies of transcriptional regulators involving ΔrpoE1, ΔphyR, ΔvjbR, and ΔblxR (ΔbabR) Brucella strains. Additionally, deletion of BM-LOV-HK decreases virulence in mice. During C57BL/6 mouse infection, the ΔBM-LOV-HK strain had 2 logs less CFUs in the spleen 3 days postinfection, but similar levels 6 days post infection compared to wild type. Infection of IRF-1(-/-) mice more specifically define ΔBM-LOV-HK strain attenuation with fewer bacteria in spleens and significantly increased survival of mutant vs. wild-type infected IRF-1(-/-) mice. Upregulation of flagella, QS, and VirB genes, along with downregulation of rpoE1 and related sigma factor, rpoH2 (BMEI0280) suggest that BM-LOV-HK modulates both QS and general stress response regulatory components to control Brucella gene expression on a global level.

Administration of a triple versus a standard double antimicrobial regimen for human brucellosis more efficiently eliminates bacterial DNA load.

The effects of doxycycline-streptomycin-rifampin versus a standard doxycycline-streptomycin regimen on residual Brucella DNA were compared in 36 acute brucellosis patients. At admission, all patients given triple (n = 22) and double (n = 14) regimens had detectable Brucella DNA with similar mean loads (P = 0.982). At follow-up, 14 to 20 months postpresentation, significantly more patients receiving triple than double regimens had undetectable Brucella DNA (P = 0.026). The doxycycline-streptomycin-rifampin regimen eliminates Brucella DNA more efficiently than doxycycline-streptomycin, which may result in superior long-term clearance of Brucella.

Replication of Brucella abortus and Brucella melitensis in fibroblasts does not require Atg5-dependent macroautophagy.

BACKGROUND: Several intracellular bacterial pathogens have evolved subtle strategies to subvert vesicular trafficking pathways of their host cells to avoid killing and to replicate inside the cells. Brucellae are Gram-negative facultative intracellular bacteria that are responsible for brucellosis, a worldwide extended chronic zoonosis. Following invasion, Brucella abortus is found in a vacuole that interacts first with various endosomal compartments and then with endoplasmic reticulum sub-compartments. Brucella establishes its replication niche in ER-derived vesicles. In the past, it has been proposed that B. abortus passed through the macroautophagy pathway before reaching its niche of replication. However, recent experiments provided evidence that the classical macroautophagy pathway was not involved in the intracellular trafficking and the replication of B. abortus in bone marrow-derived macrophages and in HeLa cells. In contrast, another study showed that macroautophagy favoured the survival and the replication of Brucella melitensis in infected RAW264.7 macrophages. This raises the possibility that B. abortus and B. melitensis followed different intracellular pathways before replicating. In the present work, we have addressed this issue by comparing the replication rate of B. abortus and B. melitensis in embryonic fibroblasts derived from wild-type and Atg5-/- mice, Atg5 being a core component of the canonical macroautophagic pathway. RESULTS: Our results indicate that both B. abortus S2308 and B. melitensis 16M strains are able to invade and replicate in Atg5-deficient fibroblasts, suggesting that the canonical Atg5-dependent macroautophagic pathway is dispensable for Brucella replication. The number of viable bacteria was even slightly higher in Atg5-/- fibroblasts than in wild-type fibroblasts. This increase could be due to a more efficient uptake or to a better survival rate of bacteria before the beginning of the replication in Atg5-deficient cells as compared to wild-type cells. Moreover, our data show that the infection with B. abortus or with B. melitensis does not stimulate neither the conversion of LC3-I to LC3-II nor the membrane recruitment of LC3 onto the BCV. CONCLUSION: Our study suggests that like Brucella abortus, Brucella melitensis does not subvert the canonical macroautophagy to reach its replicative niche or to stimulate its replication.

Splenic abscess due to brucellosis: a case report and a review of the literature.

Splenic abscess due to acute brucellosis is a rare event. We report a case of multiple splenic abscesses caused by Brucella melitensis in a 45-year-old woman and review the English language literature based on a PubMed/MEDLINE search of the last 50 years. The majority of the cases published in the literature were due to B. melitensis and a splenectomy was required in half of the cases. Antibiotics alone without surgical intervention can be successful in the treatment of patients with splenic brucellosis in the early stages of the disease.

Replication of Brucella melitensis inside primary human monocytes depends on mitogen activated protein kinase signaling.

The clinical course of infections caused by Brucella is linked to its capacity to modulate the initial immune response of macrophages in order to ensure its intracellular replication. Signal transduction pathways implicated in the survival of Brucella in human cells are not completely elucidated. We herein investigated the involvement of the TLR-MAPK-dependent signaling pathways in the survival of Brucella in primary human monocytes using live clinical strains of Brucella melitensis. B. melitensis caused a delayed, TLR2 dependent MAPK activation. Specific MAPK inhibitors for p38 (SB203580), ERK1/2 (PD98059) and JNK (SP600125) or the anti-TLR2 blocking Ab inhibited both inflammatory and anti-inflammatory responses characterized by TNF-α, IL-6 and IL-10 production. Intracellular replication of B. melitensis was mainly dependent on p38 and JNK activation and not affected by IL-10 levels. These are the first evidence to support that survival of B. melitensis inside human monocytes depends on interplay among the different MAPK family members, activated through TLR2, in spite of an initial pro-inflammatory response.

Pathogenic brucellae replicate in human trophoblasts.

Brucellae replicate in a vacuole derived from the endoplasmic reticulum (ER) in epithelial cells, macrophages, and dendritic cells. In animals, trophoblasts are also key cellular targets where brucellae efficiently replicate in association with the ER. Therefore, we investigated the ability of Brucella spp. to infect human trophoblasts using both immortalized and primary trophoblasts. Brucella extensively proliferated within different subpopulations of trophoblasts, suggesting that they constitute an important niche in cases where the fetal-maternal barrier is breached. In extravillous trophoblasts (EVTs), B. abortus and B. suis replicated within single-membrane acidic lysosomal membrane-associated protein 1-positive inclusions, whereas B. melitensis replicated in the ER-derived compartment. Furthermore, B. melitensis but not B. abortus nor B. suis interfered with the invasive capacity of EVT-like cells in vitro. Because EVTs are essential for implantation during early stages of pregnancy, the nature of the replication niche may have a central role during Brucella-associated abortion in infected women.

Autophagy favors Brucella melitensis survival in infected macrophages.

This study investigated the role of autophagy in the survival of the invasive bacterium Brucella melitensis strain 16M in murine macrophages. Here, Brucella melitensis 16M was found to trigger autophagosome formation, enhance autophagy flux and increase the expression level of the autophagy marker protein LC3-II. When autophagy was pharmacologically inhibited by 3-methyladenine (3-MA), Brucella replication efficiency was significantly decreased (p < 0.05). These results suggest that autophagy favors Brucella melitensis 16M survival in murine macrophages.

Brucella melitensis survival during manufacture of ripened goat cheese at two temperatures.

The aim of the current work was to assess the influence of two temperatures, 4°C and 24°C, on pH and water activity and their association with Brucella melitensis survival during the traditional manufacture of ripened goat cheese. Raw milk from a brucellosis-free goat herd was used for the manufacture of ripened cheese. The cheese was inoculated with 5×10(9) of the B. melitensis 16M strain during the tempering stage. The cheeses were matured for 5, 20, and 50 days at both temperatures. To assess Brucella survival, the pH and a(w) were recorded at each stage of the process (curd cutting, draining whey, immersion in brine, ripening I, ripening II, and ripening III). B. melitensis was detected at ripening stage III (1×10(3) colony-forming unit [CFU]/mL) from cheeses matured at 4°C with a pH of 5.0 and a(w) of 0.90, and at a ripening stage II (1×10(4) CFU/mL) from cheeses ripened at 24°C with a pH of 4.0 and a(w) of 0.89. The remaining stages were free from the inoculated pathogen. In addition, viable B. melitensis was recovered in significant amounts (1-2×10(6) CFU/mL) from the whey fractions of both types of cheese ripened at 24°C and 4°C. These results revealed the effects of high temperature (24°C vs. 4°C) on the low pH (4) and a(w) (0.89) that appeared to be associated with the suppression of B. melitensis at the early stages of cheese ripening. In the ripened goat cheeses, B. melitensis survived under a precise combination of temperature during maturation, ripening time, and a(w) in the manufacturing process.

Attenuation of defined Brucella melitensis wboA mutants.

Rough Brucella mutants have been sought as vaccine candidates because they do not induce seroconversion. In this study, two defined nonreverting rough mutants were derived from virulent Brucella melitensis strain 16M: a wboA deletion mutant designated WRR51 and a wboA purEK dual deletion mutant designated WRRP1. Strain WRRP1 exhibited reduced survival in human monocyte-derived macrophages (hMDMs) compared with parent strain WRR51 or with ΔpurEK strain WR201. Strain WRRP1 persisted for 1 week or less in BALB/c mice after intraperitoneal infection, while less severe attenuation was exhibited by the two single mutants in this model. Trans complementation of wboA restored the survival of WRR51 in hMDMs comparable to strain 16M and the survival of WRRP1 comparable to strain WR201.