A Brucella melitensis H38ΔwbkF rough mutant protects against Brucella ovis in rams.

Brucella melitensis and Brucella ovis are gram-negative pathogens of sheep that cause severe economic losses and, although B. ovis is non-zoonotic, B. melitensis is the main cause of human brucellosis. B. melitensis carries a smooth (S) lipopolysaccharide (LPS) with an N-formyl-perosamine O-polysaccharide (O-PS) that is absent in the rough LPS of B. ovis. Their control and eradication require vaccination, but B. melitensis Rev 1, the only vaccine available, triggers anti-O-PS antibodies that interfere in the S-brucellae serodiagnosis. Since eradication and serological surveillance of the zoonotic species are priorities, Rev 1 is banned once B. melitensis is eradicated or where it never existed, hampering B. ovis control and eradication. To develop a B. ovis specific vaccine, we investigated three Brucella live vaccine candidates lacking N-formyl-perosamine O-PS: Bov::CAΔwadB (CO2-independent B. ovis with truncated LPS core oligosaccharide); Rev1::wbdRΔwbkC (carrying N-acetylated O-PS); and H38ΔwbkF (B. melitensis rough mutant with intact LPS core). After confirming their attenuation and protection against B. ovis in mice, were tested in rams for efficacy. H38ΔwbkF yielded similar protection to Rev 1 against B. ovis but Bov::CAΔwadB and Rev1::wbdRΔwbkC conferred no or poor protection, respectively. All H38ΔwbkF vaccinated rams developed a protracted antibody response in ELISA and immunoprecipitation B. ovis diagnostic tests. In contrast, all remained negative in Rose Bengal and complement fixation tests used routinely for B. melitensis diagnosis, though some became positive in S-LPS ELISA owing to LPS core epitope reactivity. Thus, H38ΔwbkF is an interesting candidate for the immunoprophylaxis of B. ovis in B. melitensis-free areas.

Relationship between azithromycin susceptibility and administration efficacy for nontypeable Haemophilus influenzae respiratory infection.

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen that is an important cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). COPD is an inflammatory disease of the airways, and exacerbations are acute inflammatory events superimposed on this background of chronic inflammation. Azithromycin (AZM) is a macrolide antibiotic with antibacterial and anti-inflammatory properties and a clinically proven potential for AECOPD prevention and management. Relationships between AZM efficacy and resistance by NTHI and between bactericidal and immunomodulatory effects on NTHI respiratory infection have not been addressed. In this study, we employed two pathogenic NTHI strains with different AZM susceptibilities (NTHI 375 [AZM susceptible] and NTHI 353 [AZM resistant]) to evaluate the prophylactic and therapeutic effects of AZM on the NTHI-host interplay. At the cellular level, AZM was bactericidal toward intracellular NTHI inside alveolar and bronchial epithelia and alveolar macrophages, and it enhanced NTHI phagocytosis by the latter cell type. These effects correlated with the strain MIC of AZM and the antibiotic dose. Additionally, the effect of AZM on NTHI infection was assessed in a mouse model of pulmonary infection. AZM showed both preventive and therapeutic efficacies by lowering NTHI 375 bacterial counts in lungs and bronchoalveolar lavage fluid (BALF) and by reducing histopathological inflammatory lesions in the upper and lower airways of mice. Conversely, AZM did not reduce bacterial loads in animals infected with NTHI 353, in which case a milder anti-inflammatory effect was also observed. Together, the results of this work link the bactericidal and anti-inflammatory effects of AZM and frame the efficacy of this antibiotic against NTHI respiratory infection.

In vivo monitoring of Staphylococcus aureus biofilm infections and antimicrobial therapy by [18F]fluoro-deoxyglucose-MicroPET in a mouse model.

A mouse model was developed for in vivo monitoring of infection and the effect of antimicrobial treatment against Staphylococcus aureus biofilms, using the [(18)F]fluoro-deoxyglucose-MicroPET ([(18)F]FDG-MicroPET) image technique. In the model, sealed Vialon catheters were briefly precolonized with S. aureus strains ATCC 15981 or V329, which differ in cytotoxic properties and biofilm matrix composition. After subcutaneous implantation of catheters in mice, the S. aureus strain differences found in bacterial counts and the inflammatory reaction triggered were detected by the regular bacteriological and histological procedures and also by [(18)F]FDG-MicroPET image signal intensity determinations in the infection area and regional lymph node. Moreover, [(18)F]FDG-MicroPET imaging allowed the monitoring of the rifampin treatment effect, identifying the periods of controlled infection and those of reactivated infection due to the appearance of bacteria naturally resistant to rifampin. Overall, the mouse model developed may be useful for noninvasive in vivo determinations in studies on S. aureus biofilm infections and assessment of new therapeutic approaches.

Brucella ceti infection in dolphins from the Western Mediterranean sea.

BACKGROUND: Brucella ceti infections have been increasingly reported in cetaceans. Brucellosis in these animals is associated with meningoencephalitis, abortion, discospondylitis', subcutaneous abscesses, endometritis and other pathological conditions B. ceti infections have been frequently described in dolphins from both, the Atlantic and Pacific Oceans. In the Mediterranean Sea, only two reports have been made: one from the Italian Tyrrhenian Sea and the other from the Adriatic Sea. RESULTS: We describe the clinical and pathological features of three cases of B. ceti infections in three dolphins stranded in the Mediterranean Catalonian coast. One striped dolphin had neurobrucellosis, showing lethargy, incoordination and lateral swimming due to meningoencephalitis, A B. ceti infected bottlenose dolphin had discospondylitis, and another striped dolphin did not show clinical signs or lesions related to Brucella infection. A detailed characterization of the three B. ceti isolates was performed by bacteriological, molecular, protein and fatty acid analyses. CONCLUSIONS: All the B. ceti strains originating from Mediterranean dolphins cluster together in a distinct phylogenetic clade, close to that formed by B. ceti isolates from dolphins inhabiting the Atlantic Ocean. Our study confirms the severity of pathological signs in stranded dolphins and the relevance of B. ceti as a pathogen in the Mediterranean Sea.

Relative contributions of lipooligosaccharide inner and outer core modifications to nontypeable Haemophilus influenzae pathogenesis.

Nontypeable Haemophilus influenzae (NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show that opsX, lgtF, lpsA, lic1, and lic2A contribute to bacterial resistance to AMP; lic1 is related to NTHi self-aggregation; lgtF, lic1, and siaB are involved in biofilm growth; opsX and lgtF participate in epithelial infection; and opsX, lgtF, and lpsA contribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.

Spatial distribution and risk factors of Brucellosis in Iberian wild ungulates.

BACKGROUND: The role of wildlife as a brucellosis reservoir for humans and domestic livestock remains to be properly established. The aim of this work was to determine the aetiology, apparent prevalence, spatial distribution and risk factors for brucellosis transmission in several Iberian wild ungulates. METHODS: A multi-species indirect immunosorbent assay (iELISA) using Brucella S-LPS antigen was developed. In several regions having brucellosis in livestock, individual serum samples were taken between 1999 and 2009 from 2,579 wild bovids, 6,448 wild cervids and4,454 Eurasian wild boar (Sus scrofa), and tested to assess brucellosis apparent prevalence. Strains isolated from wild boar were characterized to identify the presence of markers shared with the strains isolated from domestic pigs. RESULTS: Mean apparent prevalence below 0.5% was identified in chamois (Rupicapra pyrenaica), Iberian wild goat (Capra pyrenaica), and red deer (Cervus elaphus). Roe deer (Capreolus capreolus), fallow deer (Dama dama), mouflon (Ovis aries) and Barbary sheep (Ammotragus lervia) tested were seronegative. Only one red deer and one Iberian wild goat resulted positive in culture, isolating B. abortus biovar 1 and B. melitensis biovar 1, respectively. Apparent prevalence in wild boar ranged from 25% to 46% in the different regions studied, with the highest figures detected in South-Central Spain. The probability of wild boar being positive in the iELISA was also affected by age, age-by-sex interaction, sampling month, and the density of outdoor domestic pigs. A total of 104 bacterial isolates were obtained from wild boar, being all identified as B. suis biovar 2. DNA polymorphisms were similar to those found in domestic pigs. CONCLUSIONS: In conclusion, brucellosis in wild boar is widespread in the Iberian Peninsula, thus representing an important threat for domestic pigs. By contrast, wild ruminants were not identified as a significant brucellosis reservoir for livestock.

Resveratrol therapeutics combines both antimicrobial and immunomodulatory properties against respiratory infection by nontypeable Haemophilus influenzae.

The respiratory pathogen nontypeable Haemophilus influenzae (NTHi) is an important cause of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) that requires efficient treatments. A previous screening for host genes differentially expressed upon NTHi infection identified sirtuin-1, which encodes a NAD-dependent deacetylase protective against emphysema and is activated by resveratrol. This polyphenol concomitantly reduces NTHi viability, therefore highlighting its therapeutic potential against NTHi infection at the COPD airway. In this study, resveratrol antimicrobial effect on NTHi was shown to be bacteriostatic and did not induce resistance development in vitro. Analysis of modulatory properties on the NTHi-host airway epithelial interplay showed that resveratrol modulates bacterial invasion but not subcellular location, reduces inflammation without targeting phosphodiesterase 4B gene expression, and dampens ß defensin-2 gene expression in infected cells. Moreover, resveratrol therapeutics against NTHi was evaluated in vivo on mouse respiratory and zebrafish septicemia infection model systems, showing to decrease NTHi viability in a dose-dependent manner and reduce airway inflammation upon infection, and to have a significant bacterial clearing effect without signs of host toxicity, respectively. This study presents resveratrol as a therapeutic of particular translational significance due to the attractiveness of targeting both infection and overactive inflammation at the COPD airway.

Inactivation of the Thymidylate Synthase thyA in Non-typeable Haemophilus influenzae Modulates Antibiotic Resistance and Has a Strong Impact on Its Interplay with the Host Airways.

Antibacterial treatment with cotrimoxazol (TxS), a combination of trimethoprim and sulfamethoxazole, generates resistance by, among others, acquisition of thymidine auxotrophy associated with mutations in the thymidylate synthase gene thyA, which can modify the biology of infection. The opportunistic pathogen non-typeable Haemophilus influenzae (NTHi) is frequently encountered in the lower airways of chronic obstructive pulmonary disease (COPD) patients, and associated with acute exacerbation of COPD symptoms. Increasing resistance of NTHi to TxS limits its suitability as initial antibacterial against COPD exacerbation, although its relationship with thymidine auxotrophy is unknown. In this study, the analysis of 2,542 NTHi isolates recovered at Bellvitge University Hospital (Spain) in the period 2010-2014 revealed 119 strains forming slow-growing colonies on the thymidine low concentration medium Mueller Hinton Fastidious, including one strain isolated from a COPD patient undergoing TxS therapy that was a reversible thymidine auxotroph. To assess the impact of thymidine auxotrophy in the NTHi-host interplay during respiratory infection, thyA mutants were generated in both the clinical isolate NTHi375 and the reference strain RdKW20. Inactivation of the thyA gene increased TxS resistance, but also promoted morphological changes consistent with elongation and impaired bacterial division, which altered H. influenzae self-aggregation, phosphorylcholine level, C3b deposition, and airway epithelial infection patterns. Availability of external thymidine contributed to overcome such auxotrophy and TxS effect, potentially facilitated by the nucleoside transporter nupC. Although, thyA inactivation resulted in bacterial attenuation in a lung infection mouse model, it also rendered a lower clearance upon a TxS challenge in vivo. Thus, our results show that thymidine auxotrophy modulates both the NTHi host airway interplay and antibiotic resistance, which should be considered at the clinical setting for the consequences of TxS administration.

Conjunctival vaccination against Brucella ovis in mice with mannosylated nanoparticles.

The use of sub-unit vaccines can solve some drawbacks associated with traditional attenuated or inactivated ones. However, in order to improve their immunogenicity, these vaccines needs to be associated to an appropriate adjuvant which, adequately selected, may also offer an alternative pathway for administration. The aim of this work was to evaluate the protection offered by the hot saline complex extracted from Brucella ovis (HS) encapsulated in mannosylated nanoparticles (MAN-NP-HS) when instilled conjunctivally in mice. Nanoparticles displayed a size of 300 nm and the antigen loading was close to 30 µg per mg nanoparticle. Importantly, encapsulated HS maintained its protein profile, structural integrity and antigenicity during and after the preparative process of nanoparticles. The ocular immunization was performed on BALB/c mice. Eight weeks after vaccination animals were challenged with B. ovis, and 3 weeks later, were slaughtered for bacteriological examinations. Animals immunized with MAN-NP-HS displayed a 3-log reduction in spleen CFU compared with unvaccinated animals. This degree of protection was significantly higher than that observed for the commercial vaccine (Rev1) subcutaneously administered. Interestingly, the mucosal IgA response induced by MAN-NP-HS was found to be much more intense than that offered by Rev1 and prolonged in time. Furthermore, the elicited IL-2, IL-4 and γ-IFN levels showed good correlation with the degree of protection. On the other hand, biodistribution studies in animals were performed with nanoparticles labelled with either (99m)technetium or rhodamine B isothiocyanate. The biodistribution revealed that, after instillation, MAN-NP-HS moved from the palpebral area to the nasal region and, the gastrointestinal tract. This profile of distribution was different to that observed for free (99m)TcO4⁻ colloids, which remained for at least 24h in the site of administration. In summary, mannosylated nanoparticles appear to be a safe and suitable adjuvant for conjunctival vaccination.

Determination of the viability of Toxoplasma gondii in cured ham using bioassay: influence of technological processing and food safety implications.

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii and distributed worldwide. Ingestion of viable cysts from infected raw or undercooked meat is an important route of horizontal transmission of the parasite to humans. Little information is available concerning the effect of commercial curing on cysts of T. gondii. This study is the first in which the influence of processing of cured ham on the viability of T. gondii has been evaluated, using bioassay to assess the risk of infection from eating this meat product. Naturally infected pigs were selected for the study, and a mouse concentration bioassay technique was used to demonstrate viable bradyzoites of T. gondii in porcine tissues and hams. No viable parasites were found in the final product (14 months of curing) based on results of the indirect immunofluorescence assay and histological and PCR analyses. Our results indicate that the consumption of hams cured as described here poses an insignificant risk of acquiring toxoplasmosis. However, additional studies are required to evaluate the safety of ham products cured under different conditions of curing time, salt, and nitrite concentration.

Evaluation of particulate acellular vaccines against Brucella ovis infection in rams.

The attenuated Brucella melitensis Rev 1 vaccine, used against brucellosis infection, interferes with serological diagnosis tests, may induce abortions in pregnant animals, and may infect humans. In order to overcome these drawbacks, we developed acellular vaccines based on a Brucella ovis antigenic complex (HS) containing outer membrane proteins and R-LPS entrapped in poly(anhydride) conventional and mannosylated nanoparticles (NP-HS and MAN-NP-HS) or in poly(epsilon-caprolactone) microparticles (HS-PEC) as antigen delivery systems and immunoadjuvants. Brucellosis free rams were vaccinated subcutaneously with a single dose of particles containing 3mg of HS, and challenged 6 months thereafter. Protection was evaluated by clinical, bacteriological and serological examinations, in comparison with non-vaccinated control rams. HS-PEC vaccine induced protection (7 out of 13 animals were infected) equivalent to that induced by the reference Rev 1 vaccine (8/14). In contrast, animals immunized with NP-HS were not protected, showing similar results to that obtained in the control unvaccinated rams. Furthermore HS-PEC vaccine did not interfere against B. melitensis serodiagnostic tests. In summary, HS-PEC microparticles could be used as a safe and effective vaccine against brucellosis in rams.

Rough mutants defective in core and O-polysaccharide synthesis and export induce antibodies reacting in an indirect ELISA with smooth lipopolysaccharide and are less effective than Rev 1 vaccine against Brucella melitensis infection of sheep.

Classical brucellosis vaccines induce antibodies to the O-polysaccharide section of the lipopolysaccharide that interfere in serodiagnosis. Brucella rough (R) mutants lack the O-polysaccharide but their usefulness as vaccines is controversial. Here, Brucella melitensis R mutants in all main lipopolysaccharide biosynthetic pathways were evaluated in sheep in comparison with the reference B. melitensis Rev 1 vaccine. In a first experiment, these mutants were tested for ability to induce anti-O-polysaccharide antibodies, persistence and spread through target organs, and innocuousness. Using the data obtained and those of genetic studies, three candidates were selected and tested for efficacy as vaccines against a challenge infecting 100% of unvaccinated ewes. Protection by R vaccines was 54% or less whereas Rev 1 afforded 100% protection. One-third of R mutant vaccinated ewes became positive in an enzyme-linked immunosorbent assay with smooth lipopolysaccharide due to the core epitopes remaining in the mutated lipopolysaccharide. We conclude that R vaccines interfere in lipopolysaccharide immunosorbent assays and are less effective than Rev 1 against B. melitensis infection of sheep.

Immunopathological responses and kinetics of Brucella melitensis Rev 1 infection after subcutaneous or conjunctival vaccination in rams.

The innocuousness of the Brucella melitensis Rev 1 live attenuated vaccine strain has never been fully assessed in rams. The immunopathological responses and the kinetics and distribution of the infection induced by this strain were determined after subcutaneous or conjunctival vaccination in both young (3-4 months old) and adult (12 months old) rams. At regular intervals after vaccination the animals were bled for serological studies, and slaughtered for both pathological and bacteriological examinations. The serological response after conjunctival inoculation was of lower intensity and duration than that induced subcutaneously, being the differences more evident in young rams. No genital lesions were produced and genital organs and accessory sexual glands were never found infected, being Rev 1 infection restricted to lymph nodes and spleen. Immunostained Rev 1 bacteria were located intracellularly in plasmablasts, dendritic follicular cells and macrophages in the target lymph nodes, in which cellular hyperplasia was the dominant pathological response. Subcutaneous vaccination induced a generalized infection by 2 weeks after vaccination, being then restricted to the prescapular target lymph node. Infection after conjunctival vaccination was less generalized, being restricted essentially to the cranial lymph nodes. Rev 1 infection was fully cleared by 3 months after vaccination in all animals. These results confirm the innocuousness of B. melitensis Rev 1 vaccine in rams.

Brucella outer membrane complex-loaded microparticles as a vaccine against Brucella ovis in rams.

Due to the important drawbacks of the Brucella melitensis Rev 1 vaccine, a safer vaccine based on an outer membrane complex from Brucella ovis encapsulated in poly-epsilon-caprolactone (PEC) microparticles (MP) was developed and tested in rams. Homogeneous batches of microparticles were prepared by a new double emulsion solvent evaporation method called "Total Recirculation One-Machine System" (TROMS). Such microparticles presented a mean diameter of 2 microm and displayed an antigen loading of about 13 microg HS per mg of microparticles. Subcutaneous vaccination of rams with 800 microg HS (hot saline antigenic extract of B. ovis) in PEC microparticles induced an adequate serological response against B. ovis antigens and conferred similar protection against challenge with B. ovis to that induced by the living attenuated B. melitensis Rev 1 reference vaccine. By contrast, lower doses (80 microg) of HS-PEC evoked reduced serological responses against B. ovis antigens and did not induce significant protection. The revaccination with 800 microg of HS-PEC increased the intensity and duration of the serological response against B. ovis antigens but did not improve the protection conferred by the single vaccination. Sample sera taken from any of the animals immunized with Rev 1 were seropositive in both Rose Bengal and the Complement Fixation tests (RBT, CFT) used for the diagnosis of smooth Brucella infections. By contrast, no positive reactors in both tests were recorded in the animals vaccinated with HS-PEC, being this a target objective of this study. HS-PEC microparticles can be used as a safe vaccine against brucellosis in rams, but further studies using higher doses of antigens are necessary to exploit their full potential for the prophylaxis of brucellosis in sheep.