Genotyping and phylogenetic analysis of Coxiella burnetii in domestic ruminant and clinical samples in Iran: insights into Q fever epidemiology.

Coxiella burnetii, a zoonotic pathogen, is the causative agent of Q fever, an endemic disease in Iran. However, there is currently a lack of available data on the genotypes of C. burnetii in the country. Here, we typed 26 C. burnetii isolates detected in milk, abortion, cotylodon, and cardiac valve samples from various geographical areas and hosts (7 cattle, 8 goats, 10 sheep, and 1 human) using Multilocus Variable Number Tandem Repeat Analysis (MLVA/VNTR) with five loci:ms24, ms27, ms28, ms33, and ms34. As IS1111 was observed to be spontaneously inserted in locus ms23 across all of our examined C. burnetii samples, five loci were employed for MLVA/VNTR genotyping. Among the 26 C. burnetii strains, 22 distinct genotypes (A-V) were identified in the discriminative loci. In silico analysis categorized Iranian C. burnetii strains into five genomic groups along with seven singletons, representing 11 exiting clonal complexes worldwide. Clusters 10 and 11 exclusively consisted of Iranian samples. These findings revealed high genotyping diversity among C. burnetii isolates in Iran. The genotypes circulating in Iran differed significantly from those found in other regions worldwide. To gain a comprehensive understanding of Q fever epidemiology in Iran, it is crucial to conduct large-scale studies that assess the distribution of C. burnetii genotypes across different geographical areas, hosts, and sources.

Genetic Diversity of Coxiella burnetii in Iran by Multi-Spacer Sequence Typing.

Coxiella burnetii, the zoonotic agent of Q fever, has a worldwide distribution including Iran. However, no information regarding the circulating genotype of this infection has been reported in Iran. This study aimed to evaluate the genetic diversity of C. burnetii in Iran using the multi-spacer sequence typing (MST) method. First, 14 positive C. burnetii samples (collected from four sheep, three goats, and seven cattle) were confirmed using quantitative polymerase chain reaction (qPCR) targeting the IS1111 gene. Then, ten spacers (Cox 2, 5, 18, 20, 22, 37, 51, 56, 57, and 61) were amplified using PCR for future MST analysis. The in-silico MST genotyping analysis of domestic ruminant samples revealed two new alleles (Cox5.11 and Cox56.15) in Cox5 and Cox56 loci that led to the emergence of four novel MST genotypes (MST62, 63, 64, and 65) and one MST genotype that has been previously described (MST61). This study showed the circulation of five MST C. burnetii genotypes among Iranian domestic ruminants. Understanding the C. burnetii genotypic profiles is critical in determining and preventing Q fever outbreaks.

Design of an optical nanobiosensor for detection of Legionella pneumophila in water samples.

Background and Objectives: Legionella spp. is a causative agent of Legionnaires' disease that creates public health problems. Isolation of these bacteria from water sources is essential to identify outbreak origins and prevent disease. Diagnostic biosensors for water quality control to protect consumers from water-borne infections can predict many outbreaks. Gold nanoparticles conjugated probes are a new generation of diagnostic tools. In this study, an optical nano biosensor was designed and characterized to detect Legionella pneumophila in water samples rapidly. Materials and Methods: Thiolated probes designed for the mip gene were attached to gold nanoparticles and then water samples containing Legionella pneumophila were examined. Results: The limit of detection for PCR and biosensor was 104 and 103 copy numbers/µl, respectively. Biosensor sensitivity and PCR were reported to be 90% (18 out of 20) and 85% (17 out of 20), respectively. Specificity 100% has been reported for both methods. Conclusion: According to the obtained results, this method has the potential to diagnose L. pneumophila with high sensitivity and specificity. This system can be employed as a practical tool for rapid, accurate, high-sensitivity, and acceptable detection of Legionella pneumophila in contaminated water, which is cost-effective in terms of cost and time.

Phenotypical and Genotypical Comparison of Clostridium difficile Isolated from Clinical Samples: Homebrew DNA Fingerprinting versus Antibiotic Susceptibility Testing (AST) and Clostridial Toxin Genes.

BACKGROUND: Clostridium (Clostridioides) difficile is recognized as the major cause of healthcare antibiotic-associated diarrhea. We surveyed a molecular epidemiological correlation between the clinical isolates from two general hospitals in Iran through clustering toxigenic types and antibiotic susceptibility testing (AST) accuracy. METHODS: Study population included 460 diarrhoeic specimens from inpatients with a history of antibiotic therapy. All samples underwent enriched anaerobic culture, confirmed by detection of gluD gene with PCR. Toxin status and AST were assessed by the disk diffusion method (DDM) and minimal inhibitory concentrations (MICs) of metronidazole, vancomycin, and rifampin. C. difficile outbreak was analyzed through conventional PCR by tracing toxin genes and Homebrew pulsed-field gel electrophoresis (PFGE) for characterizing isolates within our healthcare systems. RESULTS: A total of 29 C. difficile strains were isolated by enriched anaerobic culture from the clinical samples. Among them, 22 (4.8%) toxigenic profiles yielded toxins A and B (tcdA, tcdB) and binary toxins (cdtA, cdtB). The minimum inhibitory concentration (MIC) was 18.1% and 9% for vancomycin and metronidazole, and all isolates were susceptible to rifampicin and its minimum inhibitory concentration was at <0.003 µg/mL. The most dominant toxigenic and antibiotic-resistant "pulsotype F" was detected through PFGE combined with multiple Clostridial toxigenic pattern and AST. CONCLUSIONS: DNA fingerprinting studies represent a powerful tool in surveying hypervirulent C. difficile strains in clinical settings. Resistance to vancomycin and metronidazole, as first-line antibiotics, necessitate accomplishment of proper control strategies and also prescription of tigecycline as a more appropriate option.

Toxigenic Clostridium difficile-mediated diarrhoea in hematopoietic stem cell transplantation in-patients: rapid diagnosis and efficient treatment.

Allogenic hematopoietic stem cell transplant (HSCT) recipients are susceptible to any kind of infectious agents including Clostridium difficile. We studied 86 allogenic-HSCT patients who faced diarrhoea while receiving antibiotics. DNA from stool samples were explored for the presence of C. difficile toxin genes (tcdA; tcdB) by multiplex real-time PCR. Results showed nine toxigenic C. difficile amongst which seven were positive for both toxins and two were positive for tcdB. Six of toxigenic C. difficile organisms harbouring both toxin genes were also isolated by toxigenic culture. Clostridium difficile infection was controlled successfully with oral Metronidazole and Vancomycin in the confirmed infected patients.

Molecular detection of Coxiella burnetii infection in aborted samples of domestic ruminants in Iran.

BACKGROUND: Coxiella burnetii is the causative agent of Q fever which is a highly infectious zoonotic disease. C. burnetii has become one of the most important causes of abortion in livestock, which can lead to widespread abortions in these animals. There are very limited studies on the prevalence of C. burnetii infection in cases of animal abortion in Iran. The aim of this study was to investigate the occurrence of C. burnetii in ruminant abortion samples in Iran. METHODS: Abortion samples from cattle, sheep and goats were collected from different parts of Iran and were tested using Real-time PCR targeting the IS1111 element of C. burnetii. RESULTS: In this study, 36 samples (24.7%) of the 146 collected samples were positive for C. burnetii. The prevalence of C. burnetii was 21.3% (20 of 94 samples) in sheep samples. Also, 10 of 46 cattle samples (21.7%) were positive. All six goat abortion samples were positive for C. burnetii. CONCLUSIONS: The findings of the study demonstrate that C. burnetii plays an important role in domestic ruminant abortions in Iran, suggesting that more attention should be paid to the role of C. burnetii in domestic animal abortions by veterinary organizations. The risk of transmitting the infection to humans due to abortion of animals should also be considered.

Investigation of Clostridium difficile in elderly people: A preliminary study in Iran.

Elderly people are at increased risk for infections such as with Clostridium difficile. This can colonize their gut and cause various gastro-intestinal manifestations. Our survey investigated its prevalence in a nursing home in Iran. Faecal samples were collected and tested by polymerase chain reaction for identification of A, B and binary toxin genes. From 289 samples, 42(14.5%) isolates were found. Toxin genes were positive in 19 isolates (17 AþBþ and 2 isolates ABþ). The elderly are especially at risk and great attention should be paid to contamination within their nursing homes. This is not an isolated regional problem.

Potent T-cell mediated immune response against Legionella pneumophila in mice following vaccination with detoxified lipopolysaccharide non-covalently combined with recombinant flagellin A and peptidoglycan-associated lipoprotein.

BACKGROUND: Legionella pneumophila is a Gram-negative intracellular bacterium and the cause of an atypical pneumonia in humans - legionnaire's disease. Immunological assessment of bacterial antigens clarifies the way that host may develop protection against the pathogen. Lipopolysaccharide (LPS) is the main antigen of Gram-negative bacteria but is less studied because of its carbohydrate nature. Here, we immunized mice with detoxified LPS in combination with immunogenic proteins and looked into the result of bacterial challenge. METHODS: LPS of L. pneumophila was extracted by hot phenol-water method. Purified LPS was detoxified by sodium hydroxide alkaline procedure. BALB/c mice were immunized mainly with non-covalent combination of detoxified LPS (dLPS) and either of recombinant FlaA or PAL separately. Afterwards, specific serum IgG was assessed by ELISA. Mice were challenged intravenously with sublethal dose of L. pneumpphila then splenocytes were cultured. Cytokine responses of splenocytes were analyzed by ELISA. RESULTS: Polysaccharide antigen did not elicit significant serum IgG. Combination of the dLPS with recombinant FlaA and PAL led to risen IgG and its subclasses (IgG1, IgG2a and IgG2b) against polysaccharide. Mice immunized with combination of the dLPS and recombinant proteins showed significant elevation of cytokine responses in splenocyte culture after being challenged with L. pneumophila. CONCLUSIONS: Our results suggest that combination of polysaccharide antigen derived from Legionella LPS may confer raised cell-mediated responses against the pathogen when combined with Th-1 stimulating protein antigens. Although not covalently bond, Legionella detoxified LPS combination with recombinant FlaA and PAL effectively elicited Th-1 type cytokines and humoral responses against L. pneumophila in BALB/c mice.

Nanoparticle-based immunotherapy of breast cancer using recombinant Helicobacter pylori proteins.

Breast cancer is the most common cancer in women worldwide and is associated with substantial medical and economic burden. We report the development of a hybrid immunotherapeutic system based on recombinant Nap protein from Helicobacter pylori (HP-Nap) for the treatment of breast tumors. Chitosan nanoparticles with pseudo-spherical morphology and positive zeta potential were synthesized as carriers for HP-Nap. In vitro study was performed on mouse breast cancer cell line (4T1) and human breast cancer cell lines (MCF7). In vivo study was done on 4T1 tomural mice. TUNEL assay and real time PCR test were performed on tumor mice receiving the nanoparticle treatment. The nanoparticle-protein complex induced apoptosis in vitro in cultured breast cancer cells. In-vivo studies on inbred, female BALB/c mice confirmed the shrinkage of tumor mass after administration of the nanoparticle complex containing HP-Nap. The TUNEL assay further confirmed apoptosis in extracted mouse breast cancer cells. A decrease in the expression of VEGF and MMP9 genes was observed in 4T1 cells as shown by real time PCR. Our data suggesting that the therapeutic nanocomplex may have led to decreased tumor growth in mice through changing the production rate of cytokines and increasing tumoricidal activities of the immune system.

Investigating the role of L. pnuemophila LPS derivatives in formation of specific cell-mediated immune responses against the pathogen.

Legionella pneumophila is a Gram-negative intracellular bacterium and causes legionnaire's disease an -atypical pneumonia in humans. Lipopolysaccharide (LPS) is the main antigen of Gram-negative bacteria but is less studied because of its carbohydrate nature. Here, we immunized mice with detoxified LPS and O-antigen polysaccharide in combination with bovine serum albumin (BSA) and explored the immunological responses of mice to the bacterial infection. LPS of L. pneumophila was extracted by hot phenol-water method. Purified LPS was detoxified by sodium hydroxide alkaline procedure. O-polysaccharide antigen (OPS) obtained by acetic acid treatment of LPS. BALB/c mice were immunized mainly with non-covalent combination of detoxified LPS (dLPS) or OPS with BSA separately. Pure polysaccharide antigens did not elicit significant serum IgG against LPS. Combination of the dLPS and OPS with BSA resulted in risen IgG and its subclasses (IgG1 and IgG2a) against lipopolysaccharide. Mice were challenged intravenously with sublethal dose of L. pneumpphila. Then, splenocytes were cultured and cytokine responses of splenocytes to pathogenic Legionella was studied by ELISA. Mice immunized with combination of the dLPS or OPS and BSA showed significant elevation of cytokine responses to pathogenic L. pneumophila. Our results suggest that combination of the polysaccharide antigen derived from Legionella LPS may confer raised cell-mediated responses against the pathogen when combined with a protein antigen which is capable of eliciting cell-mediated responses. Although not covalently bond, Legionella polysaccharides combined with BSA effectively elicited Th-1 type cytokines and humoral responses against L. pneumophila in BALB/c mice.

Clarithromycin-Susceptible But Virulent Helicobacter pylori Strains Infecting Iranian Patients' Stomachs.

INTRODUCTION: Helicobacter pylori was discovered first in the stomachs of patients with gastritis and ulcers by Marshall and Warren in 1982. This discovery majorly affected many research areas of gastroenterology. Since then, the main aim has been to eradicate this microaerophilic bacterium from the stomachs of infected subjects. METHODS: We studied symptomatic cases by endoscopic surgery and examined the prevalence of cagA-vacA genotypes among the H. pylori isolates. H. pylori isolated from antral biopsies of patients with gastritis and duodenal ulcer were subjected to antimicrobial susceptibility testing and PCR genotyping by using routine bacterial cultures. Clarithromycin-susceptibility profiling was done by the E-test. DNA was extracted using standard manufacturer protocols with minor modifications and cagA and vacA genotyping was done PCR. RESULTS: In our study, all strains identified as H. pylori in culture (61/81) were confirmed by PCR by amplifying a fragment of the glmM gene. Totally, 61 patients were confirmed to be positive for H. pylori and they were included in the genotyping and antibiotic-susceptibility testing. Thirteen H. pylori strains were determined to be resistant to clarithromycin. DISCUSSION: Current accumulating data indicate that both clarithromycin-resistant and susceptible isolates of H. pylori need to be screened and tracked in populations.

A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates.

BACKGROUND Clostridium difficile is the major causative agent of nosocomial antibiotic-associated colitis. The gold standard for C. difficile detection is stool culture followed by cytotoxic assay, although it is laborious and time-consuming. We developed a screening test based on a two-step conventional polymerase chain reaction (PCR) approach to detect gluD, the glutamate dehydrogenase (GDH) enzyme gene, which is a marker for screening of C. difficile. Targeting gluD comparing to the conserved stable genetic element of pathogenicity locus (PaLoc), with an accessory gene of Cdd3, was an effective method for the detection of this pathogen from patients with enterocolitis. METHODS Fresh fecal samples of the patients who were clinically suspicious for antibiotic-associated colitis were collected. Stool specimens were cultured on the cycloserine-cefoxitin fructose agar (CCFA) in an anaerobic condition, following alcohol shock treatment and enrichment in Clostridium difficile Brucella broth (CDBB). On confirmed colonies, PCR was carried out for detection of PaLoc subsidiary gene, Cdd3, and toxicogenic genes, tcdA and tcdB. The gluD that is GDH gene detection was performed by conventional PCR on the extracted DNA from 578 fresh stool samples. RESULTS 57 (9.8%) strains of C. difficile were approved by conventional PCR for gluD and Cdd3 genes, in which 37 (6.4%) colonies had tcdA+/tcdB+ genotype, 2 (0.3%) tcdA+/tcdB-, 4 (0.7%) tcdA-/ tcdB+ and the remaining 14 (2.4%) colonies were tcdA and tcdB negative. CONCLUSION These results demonstrate that targeting gluD by PCR is quite promising for rapid detection of C. difficile from fresh fecal samples. Furthermore, the multiple-gene analysis for tcdA and tcdB assay proved a reliable approach for diagnosing of toxigenic strains among clinical samples.

Expression of adhesin genes and biofilm formation among Klebsiella oxytoca clinical isolates from patients with antibiotic-associated haemorrhagic colitis.

PURPOSE: Biofilm formation and resistance to last-line antibiotics have restricted chemotherapy options toward infection eradication. METHODOLOGY: Fifty K. oxytoca isolates were collected from patients with antibiotic-associated haemorrhagic colitis (AAHC). Antibiotic susceptibility tests were conducted and phenotypic biofilm formation was assessed using microtitre tissue plate (MTP) assay. PCR was employed to amplify the adhesins, extended-spectrum ß-lactamases (ESBLs), carbapenemase and colistin resistance genes. The expression of adhesin genes was evaluated using quantitative real-time PCR (RT-qPCR).Results/Key findings. The previous antibiotic consumption and hospitalization (P<0.05) and older ages (P=0.0033) were significantly associated with AAHC. None of the isolates produced biofilm strongly, but 70% of them produced moderate-level biofilm. The blaCTX-M (12/14), the blaIMP (8/14 MICIMI =4 µg ml-1 ) and blaOXA-48-like (5/14) and mcr-1 (4/14) genes were predominant, three of which harbouring all the genes. The expression of matB (0.023) and mrkA (0.011) was significantly different between multidrug-resistant and susceptible isolates. Furthermore, moderately biofilm producer isolates significantly exhibited higher expression of fimA (P=.0117), pilQ (P=0.002) and mrkA (P=0.020) genes compared to biofilm non-producers. No significant difference regarding gene expression was observed among ESBL alleles. CONCLUSION: Bacterial attachment by adhesins and biofilm formation among extensive drug-resistant K. oxytoca isolates hinder the efficient infection eradication. Hence, control and surveillance studies should be performed and other therapeutic auspicious approaches must be taken into account against AAHC, biofilm formation and drug resistance spread. Furthermore, previous antibiotic consumption and long-term hospitalization should be controlled.

High prevalence and risk factors of Coxiella burnetii in milk of dairy animals with a history of abortion in Iran.

Coxiella burnetii is causative agent of Q fever, which is a public health problem in most countries. The aim of this study was to study the prevalence rate of C. burnetii in raw milk of dairy animals in Iran with previous history of abortion. In this survey, milk samples were collected from different dairy animals with history of abortion from Qom province (center of Iran). Samples were tested by Nested PCR and Real-time PCR for detection of IS1111 gene of C. burnetii. In total, 34.92% (44 of 126) milk samples were positive for C. burnetii. Prevalence of C. burnetii in cattle, sheep and goat milk was 33.33%, 35.71% and 35.71%, respectively. Age was a significant risk factor for shedding of C. burnetii in cattle (P = 0.02) and goat (P = 0.05). Shedding of C. burnetii was high prevalence in milk of dairy animals with history of abortion in Iran. The high prevalence of this bacterium in milk (especially in animals with history of abortion) indicates that Excreted by milk as a potential source to spread of infection in the environment.

Genetic diversity and amino acid sequence polymorphism in Helicobacter pylori CagL hypervariable motif and its association with virulence markers and gastroduodenal diseases.

Genetic variability in cagL gene especially within the Helicobacter pylori CagL hypervariable motif (CagLHM) may affect the development of gastric cancer. Therefore, this study was conducted to investigate the association of CagL diversity with clinical outcomes and with H pylori virulence markers. A total of 126 patients with different gastric diseases including non-ulcer dyspepsia (NUD), peptic ulcer disease (PUD), gastric erosion (GE), and gastric cancer (GC) were enrolled. H pylori was cultured from gastric biopsies, and the isolates were screened for the presence of cagL, cagA, vacA, babA2, sabA, and cagPAI integrity by PCR. The amino acid polymorphisms of cagL were analyzed using DNA sequencing. We isolated 61 (48.4%) H pylori strains from 36 NUD, eight PUD, 12 GE, and five GC patients. Almost all isolates were cagL positive (97%), and their RGD, RHS, and SKIIVK motifs were highly conserved. Among 10 CagLHM variants identified, NEIGQ and NKIGQ were detected as the most prevalent sequences. Interestingly, a significant association was found between the presence of NKMGK and PUD (P = 0.002). Notably, the NEIGQ isolates with multiple C-type EPIYA repeat that carried intact cagPAI correlated with disease risk for PUD, GE, and GC (P = 0.021). In conclusion, we identified novel variants of H pylori CagLHM sequences in Iranian population such as NKMGK, which was associated with disease risk for PUD. Further studies using a large number of strains are required to better clarify the function of certain CagLHM motifs in gastric carcinogenesis and disease outcome.

Molecular prevalence of Coxiella burnetii in milk in Iran: a systematic review and meta-analysis.

Q fever is a major zoonotic disease in the world. The aim of this meta-analysis was to estimate the prevalence of Coxiella burnetii in animal milk in Iran. We systematically reviewed the literature to identify eligible studies from January 2008 to June 2016 in English or Farsi (Persian) databases. We extracted the molecular prevalence of C. burnetii in milk from cows, goats, sheep, and camels in Iran. The total prevalence of C. burnetii in cow milk was 15.09% (95% CI 11.08-19.10) by PCR methods. The highest and lowest prevalence of Q fever agent were seen in the East Azerbaijan (25.55%) and Khorasan-Razavi (4.22%) provinces, respectively. The molecular prevalence of C. burnetii in goat milk was 7.80% (95% CI 3.54-12.07%). The provinces of Qom (0%) and Lorestan (44.71%) had the lowest and the highest frequency of C. burnetii infection in goat's milk, respectively. Total prevalence of C. burnetii in sheep milk was 3.79% (95% CI 0.72-6.87%). The highest frequency of C. burnetii in sheep milk was detected in the Khorasan-Razavi province (34.78%). The frequency of C. burnetii in camel milk was 1.43%. High infection of C. burnetii in milk is an important health problem in Iran, amplified by the traditional preparations of dairy products.

Genetic evidence of Coxiella burnetii infection in acute febrile illnesses in Iran.

Mounting evidence suggests that Q-fever is more prevalent in Iran than originally believed. However, in most parts of the country, clinicians do not pay enough attention to Q fever in their differential diagnosis. The aim of this study was to investigate the prevalence of Coxiella burnetii in suspected cases of acute Q fever in north-western Iran using molecular techniques. Febrile patients were enrolled in the study and investigated for C. burnetii infection. Sera samples were tested using real-time PCR for detection of IS1111 gene, and positive samples were confirmed with nested PCR. Nine patients (4.2%) out of 216 suspected cases were positive for C. burnetii. Weakness and fatigue, headache, and lethargy were the most prevalent clinical symptoms in acute Q fever patients. According to the results of this study and other reports of human cases in Iran, the diagnosis system of Q fever in Iran should be urgently revamped.

Recombinant flagellin-PAL fusion protein of Legionella pneumophila induced cell-mediated and protective immunity against bacteremia in BALB/c mice.

We report a new recombinant fusion protein composed of full-length Legionella pneumophila flagellin A and peptidoglycan-associated lipoprotein (PAL), rFLA-PAL, capable of inducing protective immunity against L. pneumophila. The recombinant protein was over expressed in Escherichia coli strain BL21 (DE3) using pET-28a (+) expression vector (pET28a-flaA-pal) and purified by Ni2+ exchange chromatography. Immunological properties of rFLA-PAL were assessed in a mouse model. Female BALB/c mice, immunized with rFLA-PAL, exhibited a rapid increase in serum antibody concentration against each of its protein portions. Furthermore, a strong activation of both innate and adaptive cell-mediated immunity was observed as indicated by antigen-specific splenocyte proliferation, IFN-γ and IL-12 production, and early production of TNF-α in the serum and in splenocyte cultures which were separately assessed against PAL and FLA. BALB/c mice were challenged with a lethal dose of L. pneumophila intravenously. In a 10-days follow-up after intravenous lethal challenge with L. pneumophila, a 100% survival rate was observed for mice immunized with rFLA-PAL, same as for those immunized with a sublethal dose of L. pneumophila. Based on the potent immune responses observed in mice immunized with rFLA-PAL, this recombinant fusion protein could be a potential vaccine candidate against the intracellular pathogen L. pneumophila.

Protection against Legionnaire's Disease: Recombinant Flagellin A of Legionella pneumophila Can Induce Protective Immunity against Bacteremia in a BALB/c Murine Model.

To investigate the immunoprotective effects of the recombinant type A flagellin (FLA), the flaA gene of Legionella pneumophila serogroup 1 strain Paris was cloned into pET28a(+). Recombinant FLA (rFLA) was overexpressed in E. coli BL21 (DE3) and purified by Ni2+ exchange chromatography. Female BALB/c aged 6-8 weeks were immunized with 20 µg of rFLA. Nonimmunized mice along with mice inoculated with a sublethal dose of live L. pneumophila intravenously were considered as negative and positive controls, respectively. A significant serum antibody response was observed in female BALB/c mice immunized with rFLA. Production of IFN-γ and IL-12, and TNF-α in the serum and the splenocyte cultures, and antigen-specific splenocyte proliferation suggested a strong innate and adaptive cell-mediated immunity response in rFLA-immunized mice. Intravenous lethal challenge with L. pneumophila serogroup 1 (strain Paris) showed that 60% of mice immunized with rFLA survived in a 10-day follow-up survey. These results show that rFLA from L. pneumophila can elicit strong innate and adaptive immune responses and suggest the possibility of a long-term immunity against lethal challenge with L. pneumophila.

Evaluation of nitric oxide production and proliferation activity of recombinant Bacterioferritin of Helicobacter pylori on macrophages.

INTRODUCTION: Helicobacter pylori is a specific pathogen found in the human stomach. The Bacterioferritin of Helicobacter pylori is a major virulence factor of this pathogen which little is known about its effect on immune system. The aim of this study is to assess the effect of recombinant Bacterioferritin Helicobacter pylori on the production of nitric oxide (NO) and the activity and viability of macrophages derived from mice peritoneal. MATERIAL AND METHOD: The Bacterioferritin protein of Helicobacter pylori was cloned and purified. Mice peritoneal macrophages were purified and cultured. Different concentrations of recombinant protein were used to stimulate macrophages which had received LPS simultaneously. Cell survival and nitric oxide (NO) production were measured subsequently. RESULTS: Our results elucidated that the recombinant protein induced a significant NO production at a dose of 30 µg/ml (P < 0.01) compared to the control which was accompanied by increasing in the viability of macrophages at dosage of 30 µg/ml. CONCLUSION: According to our findings, recombinant protein stimulates peritoneal macrophages to produce NO and does not have cytotoxic effect. Therefore, it is suggested that recombinant Bacterioferritin can be studied further as a vaccine candidate for Immunotherapy purposes.