Protective antigen domain 4 of Bacillus anthracis as a candidate for use as vaccine for anthrax.

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B. anthracis, was expressed in the bacterial system of E. coli. In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.

Detection of Coxiella burnetii and Francisella tularensis in Tissues of Wild-living Animals and in Ticks of North-west Poland.

This work presents results of the research on the occurrence of Coxiella burnetii and Francisella tularensis in the tissues of wild-living animals and ticks collected from Drawsko County, West Pomeranian Voivodeship. The real-time PCR testing for the pathogens comprised 928 samples of animal internal organs and 1551 ticks. The presence of C. burnetii was detected in 3% of wild-living animals and in 0.45-3.45% (dependent on collection areas) of ticks. The genetic sequences of F. tularensis were present in 0.49 % of ticks (only in one location - Drawa) and were not detected in animal tissues. The results indicate respectively low proportion of animals and ticks infected with C. burnetii and F. tularensis .This work presents results of the research on the occurrence of Coxiella burnetii and Francisella tularensis in the tissues of wild-living animals and ticks collected from Drawsko County, West Pomeranian Voivodeship. The real-time PCR testing for the pathogens comprised 928 samples of animal internal organs and 1551 ticks. The presence of C. burnetii was detected in 3% of wild-living animals and in 0.45­3.45% (dependent on collection areas) of ticks. The genetic sequences of F. tularensis were present in 0.49 % of ticks (only in one location ­ Drawa) and were not detected in animal tissues. The results indicate respectively low proportion of animals and ticks infected with C. burnetii and F. tularensis.

Brucella - Virulence Factors, Pathogenesis and Treatment.

Brucellae are Gram-negative, small rods infecting mammals and capable of causing disease called brucellosis. The infection results in abortion and sterility in domestic animals (sheeps, pigs, rams etc). Especially dangerous for humans are: Brucella melitensis, Brucella suis, Brucella abortus, and Brucella canis that trigger unspecific symptoms (flu-like manifestation). Brucella rods are introduced via host cells, by inhalation, skin abrasions, ingestion or mucosal membranes. The most important feature of Brucella is the ability to survive and multiply within both phagocytic and non-phagocytic cells. Brucella does not produce classical virulence factors: exotoxin, cytolisins, exoenzymes, plasmids, fimbria, and drug resistant forms. Major virulence factors are: lipopolysaccharide (LPS), T4SS secretion system and BvrR/BvrS system, which allow interaction with host cell surface, formation of an early, late BCV (Brucella Containing Vacuole) and interaction with endoplasmic reticulum (ER) when the bacteria multiply. The treatment of brucellosis is based on two-drug therapy, the most common combinations of antibiotics are: doxycycline with rifampicin or fluoroquinolones with rifampicin. Currently, also other methods are used to disrupt Brucella intracellular replication (tauroursodeoxycholic acid or ginseng saponin fraction A).

Microbiological analysis of bioaerosols collected from Hospital Emergency Departments and ambulances.

INTRODUCTION AND OBJECTIVE: The goal of the study was a microbiological, qualitative and quantitative analysis of bioaerosol at the workplace of medical personnel (Health Emergency Departments (HEDs), ambulances), and comparative administration offices with an expected neutral occupational exposure to biological agents measured with individual Button Sampler. MATERIAL AND METHODS: Personal sampling was performed with Button Sampler instrument loaded with gelatine filters in 10 HEDs, in 9 ambulances and in 9 offices to assess the occupational biological agents' exposure in air. Sampling was conducted from March until April 2016. Samples were quantitatively assessed for viable and total number of bacteria and fungi. Routine procedures for microbiological diagnostics were implemented. Data were analysed using Kruskal-Wallis and Mann-Whitney statistical tests with α=0.05. P value less than 0.05 were considered significant. RESULTS: At the workplaces assessed, the concentrations of viable microorganisms in HEDs were 1.3×102 - 4.2×103 CFU/m3 for bacteria, 3.4×100 - 8.1×101 CFU/m3 for fungi; in ambulances 1.3×102 - 1.4×103 CFU/m3 (bacteria), 6.7×100 - 6.5×102 CFU/m3 (fungi) and in offices 4.2×101 - 5.0×103 CFU/m3 (bacteria), 0 - 7.9×102 CFU/m3(fungi). In outdoor air, the number of microorganisms reached the level: 1.0×102 - 5.9×102 CFU/m3 for bacteria and 1.5×102 - 8.2×102 CFU/m3 for fungi. The predominant isolated bacteria were Gram-positive cocci. The prevalent fungi species belonged to the genus Aspergillus and Penicillium. CONCLUSIONS: The quantitative assessment of examined indoor air was similar to control outdoor air, and were relatively low. The level of microbiological contamination did not exceed 5×103 CFU/m3 which is recommended as an admissible level in public spaces in Poland.

Evaluation of the Levels and Quality of Microbial Contamination in Medical Emergency Departments in Comparison to Other Workplaces.

Work in Hospital Emergency Departments (HEDs) exposes both the emergency ward staff and patients to infectious and in other way harmful biological agents. The results of this study shows the presence of pathogenic bacteria isolated by three different methods. It revealed 9.8% of pathogens detected by imprint method, 10.5% of pathogens by swabbing method, 17.6% and 22% in HEDs corridors and rooms, respectively, by air sampling method. In control workplaces (offices) pathogenic bacteria reached the level of 6.5% and 14.7% by imprint method and swabbing, respectively. The relatively low level of contamination by bacteria in HEDs may depend on the effectiveness of Standard Protective Precautions in the studied hospitals.

Bacillus anthracis infections--new possibilities of treatment.

INTRODUCTION AND OBJECTIVE: Bacillus anthracis is one of biological agents which may be used in bioterrorism attacks. The aim of this study a review of the new treatment possibilities of anthrax, with particular emphasis on the treatment of pulmonary anthrax. Abbreviated description of the state of knowledge. Pulmonary anthrax, as the most dangerous clinical form of the disease, is also extremely difficult to treat. Recently, considerable progress in finding new drugs and suitable therapy for anthrax has been achieved, for example, new antibiotics worth to mentioning, levofloxacin, daptomycin, gatifloxacin and dalbavancin. However, alternative therapeutic options should also be considered, among them the antimicrobial peptides, characterized by lack of inducible mechanisms of pathogen resistance. Very promising research considers bacteriophages lytic enzymes against selected bacteria species, including antibiotic-resistant strains. RESULTS: Interesting results were obtained using monoclonal antibodies: raxibacumab, cAb29 or cocktails of antibodies. The application of CpG oligodeoxynucleotides to boost the immune response elicited by Anthrax Vaccine Adsorbed and CMG2 protein complexes, also produced satisfying therapy results. Furthermore, the IFN-α and IFN-ß, PA-dominant negative mutant, human inter-alpha inhibitor proteins and LF inhibitors in combination with ciprofloxacin, also showed very promising results. CONCLUSIONS: Recently, progress has been achieved in inhalation anthrax treatment. The most promising new possibilities include: new antibiotics, peptides and bacteriophages enzymes, monoclonal antibodies, antigen PA mutants, and inter alpha inhibitors applications. In the case of the possibility of bioterrorist attacks, the examination of inhalation anthrax treatment should be intensively continued.

Prevalence of Coxiella burnetii in environmental samples collected from cattle farms in Eastern and Central Poland (2011-2012).

Coxiella burnetii is the etiologic agent of Q fever. It may occur as two different morphological forms, a large cell variant (LCV) and a small cell variant (SCV). The SCV is characterized by unique resistance to physical and chemical factors and may survive in the environment for many months. The objective of this study was to examine environmental samples for the presence of C. burnetii using real-time PCR in areas where Q fever was previously reported and in randomly selected animal farms where Q fever was not reported. The samples were collected in the following provinces in Poland: Lublin, Subcarpathian and Masovian. Monitoring was performed with real-time PCR and serological methods. Of the 727 environmental samples, 33 (4.54%) contained the multi-copy insertion sequence IS1111, which is specific for C. burnetii. Subsequently, the presence of C. burnetii antibodies was determined using serological tests in selected herds in which positive genetic results were obtained. Serological analyses of 169 serum samples using CFT and ELISA were performed on Polish black-and-white Holstein-Friesian cows and one cow imported from Denmark. Using the CFT method, 11 samples were positive for phase I antibodies and six were positive for phase II antibodies. Moreover, in two cases, the presence of antibodies specific for both phase I and phase II antigens of C. burnetii was detected. However, of the 169 examined serum samples, 20 were positive by ELISA test, of which six were also positive by CFT. Additionally, multi spacer typing (MST) of isolated C. burnetii strains was performed. The MST results identified two new genotypes in Poland, ST3 and ST6. The results indicate that continued research regarding spread of this pathogen within a country is necessary.

Q fever--selected issues.

Q fever is an infectious disease of humans and animals caused by Gram-negative coccobacillus Coxiella burnetii, belonging to the Legionellales order, Coxiellaceae family. The presented study compares selected features of the bacteria genome, including chromosome and plasmids QpH1, QpRS, QpDG and QpDV. The pathomechanism of infection--starting from internalization of the bacteria to its release from infected cell are thoroughly described. The drugs of choice for the treatment of acute Q fever are tetracyclines, macrolides and quinolones. Some other antimicrobials are also active against C. burnetii, namely, telitromycines and tigecyclines (glicylcycline). Q-VAX vaccine induces strong and long-term immunity in humans. Coxevac vaccine for goat and sheep can reduce the number of infections and abortions, as well as decrease the environmental transmission of the pathogen. Using the microarrays technique, about 50 proteins has been identified which could be used in the future for the production of vaccine against Q fever. The routine method of C. burnetii culture is proliferation within cell lines; however, an artificial culture medium has recently been developed. The growth of bacteria in a reduced oxygen (2.5%) atmosphere was obtained after just 6 days. In serology, using the IF method as positive titers, the IgM antibody level >1:64 and IgG antibody level >1:256 (against II phase antigens) has been considered. In molecular diagnostics of C. burnetii infection, the most frequently used method is PCR and its modifications; namely, nested PCR and real time PCR which detect target sequences, such as htpAB and IS1111, chromosome genes (com1), genes specific for different types of plasmids and transposase genes. Although Q fever was diagnosed in Poland in 1956, the data about the occurrence of the disease are incomplete. Comprehensive studies on the current status of Q fever in Poland, with special focus on pathogen reservoirs and vectors, the sources of infection and molecular characteristics of bacteria should be conducted.

New aspects of the infection mechanisms of Bacillus anthracis.

Articles concerning new aspects of B. anthracis mechanisms of infection were reviewed. It was found, that the hair follicle plays an important role in the spore germination process. The hair follicle represent an important portal of entry in the course of the cutaneous form of disease infections. After mouse exposition to aerosol of spores prepared from B. anthracis strains, an increase in the level of TNF-α cytokines was observed. The TNF-α cytokines were produced after intrusion into the host by the microorganism. This process may play a significant role in the induced migration of infected cells APCs (Antigen Presenting Cells) via chemotactic signals to the lymph nodes. It was explained that IgG, which binds to the spore surface, activates the adaptive immune system response. As a result, the release C3b opsonin from the spore surface, and mediating of C3 protein fragments of B. anthracis spores phagocytosis by human macrophages, was observed. The genes coding germination spores protein in mutant strains of B. anthracis MIGD was a crucial discovery. According to this, it could be assumed that the activity of B. anthracis spores germination process is dependent upon the sleB, cwlJ1 and cwlJ2 genes, which code the GSLEs lithic enzymes. It was also discovered that the specific antibody for PA20, which binds to the PA20 antigenic determinant, are able to block further PA83 proteolytic fission on the surface of cells. This process neutralized PA functions and weakened the activity of free PA20, which is produced during the PA83 enzyme fission process. Interaction between PA63 monomer and LF may be helpful in the PA63 oligomerization and grouping process, and the creation of LF/PA63 complexes may be a part of an alternative process of assembling the anthrax toxin on the surface of cells. It was found that actin-dependent endocytosis plays an important role in the PA heptamerisation process and leads to blocking the toxin activity. Chaperones, a protein derived from host cells, may be helpful in ATP and cytosolic factors translocation, and in this way increase the translocation of diphteria toxin A domein (DTA) and substrate of fusion protein LF(N)-DTA.

[Selected research problems of anthrax vaccine development]. / Wybrane kierunki badan nad szczepionkami przeciwko waglikowi.

The threat of bioterrorism with B. anthracis against civilian population is one of major concern. After successful bioterroristic attack in 2001 in US renewed research interest has prompted in the development of new and more effective vaccine against anthrax. There are two licensed vaccines against anthrax--AVA-Bio-Thrax US and UK--sterile culture filtrate prepared by alum precipitation. Both vaccines are based on PA antigen. There are several concerns regarding PA based vaccines. They require six sc injections and yearly booster, high rates of local reaction after vaccination is observed, the immunity is not long lasting, vaccination do not protect animals against different strains of B. anthracis. New strategies in the development of anthrax vaccines have been presented (recombinant PA, subunits vaccine, mutants, conjugated). Using proteomic approaches new antigens have been also identified as candidates for future vaccines. More effective and easy to perform methods of vaccination have been reviewed.

Genetic diversity among Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis strains using repetitive element polymorphism-PCR.

Repetitive element polymorphism-PCR (REP-PCR) is one of the tools that has been used to elucidate genetic diversity of related microorganisms. Using the MB1 primer, REP-PCR fingerprints from 110 Bacillus strains within the "B. cereus group" have identified eighteen distinct categories, while other more distantly related bacterial species fell within six additional categories. All Bacillus anthracis strains tested were found to be monomorphic by fluorophore-enhanced REP-PCR (FERP) fingerprinting using the MB1 primer. In contrast, other non- B. anthracis isolates displayed a high degree of polymorphism. Dendrogramic analysis revealed that the non- B. anthracis strains possessing the Ba813 chromosomal marker were divided into two clusters. One of the clusters shared identity with the B. cereus strains examined.