Effect of plasma-activated water on the biofilm-forming ability of Salmonella enterica serovar Enteritidis and expression of the related genes.

In recent years, microbial decontamination with plasma-activated water (PAW) has attracted a lot of research attention in the field of food industry. Despite several studies showing that PAW effectively inactivates planktonic bacteria, few studies have been conducted on biofilms. The present study was, therefore, designed to evaluate the effect of PAW on the biofilm formation characteristics of Salmonella Enteritidis. Comparing the expression patterns of biofilm-related genes in PAW-treated and non-treated planktonic and biofilm cells provided insight into how PAW regulates this process. The results showed that a 30-minute exposure to PAW at room temperature significantly reduced S. enteritidis planktonic cells. This exposure resulted in a decreased expression of the genes involved in the early stages of biofilm formation (csgD, agfA, fimA, lpfE, and rpoS), and an increased expression of the csrA gene in S. enteritidis planktonic cells. These results indicated the inhibitory effect of PAW on the biofilm formation process in S. enteritidis. Results of the initial attachment assay confirmed these findings, where, after 6 h, the number of PAW-treated cells attached to the stainless steel surfaces were significantly lower than non-treated ones. Furthermore, biofilm development assay revealed that the number of PAW-treated biofilm cells were significantly lower than non-treated ones after 24 h incubation at 37 °C. These findings were confirmed by measurements of the major components of biofilm i.e., extracellular DNA (eDNA), protein and carbohydrate. The amount of these components in 24-hour biofilms produced by PAW-treated S. enteritidis cells was significantly lower than that of non-treated cells. PAW's treatment on preformed 24-hour biofilms for 30 min led to a decrease in the expression of genes involved in quorum sensing and cellulose synthesis (csgD, bapA, adrA, luxS and sdiA) and an increase in the expression of the csrA gene. This treatment also reduced the number and metabolic activity of biofilm cells compared to non-treated biofilm cells. In total, the present study demonstrated that PAW has an inhibitory effect on the process of biofilm formation in S. enteritidis and hence, the food industry should pay special attention to PAW as a promising treatment to eliminate bacterial biofilms.

Productivity Loss of Temporary Work Absenteeism Due to COVID-19 and Its Determinant Factors in Northeastern Iran.

OBJECTIVE: This study aimed to estimate the lost productivity cost of temporary work absenteeism due to COVID-19. METHODS: This study conducted on all hospitalized patients with COVID-19 in northeastern Iran between February 2020 and March 2022 (10,406 cases). Data were collected from the Hospital Information System. Indirect costs were estimated using the human capital approach. Data were analyzed with the STATA version 17. RESULTS: The total indirect cost of work absenteeism due to COVID-19 was estimated at $513,688. There was a statistically significant relationship between the mean lost productivity cost and COVID-19 peak, sex, insurance type, age, and hospitalization. CONCLUSIONS: Because the absenteeism costs of COVID-19 had increased in the second peak, which coincided with the summer holidays, the country's crisis management headquarters should pay more attention to formulating and implementing appropriate preventive programs in future epidemics.

Designing of novel chimeric PvpA-pMGA protein of Mycoplasma gallisepticum, applicable for indirect ELISA.

BACKGROUND: Mycoplasma gallisepticum is the primary agent of chronic respiratory disease in chickens creating important economic losses in poultry industry. pMGA and pvpA genes encode major surface proteins in M. gallisepticum containing pathogenic, antigenic, and immune evasion characteristics. The objective of the present study was to design, express, and purify the recombinant chimeric PvpA-pMGA protein from M.gallisepticum for using in serological diagnostic test. METHODS: Antigenic regions of PvpA and pMGA proteins were predicted for designing chimeric pvpA-pMGA gene construct. The codon optimized sequence was cloned into the expression vector pET32a+ and transformed into the Escherichia coli strain BL21 (DE3). The pET32a-PvpA-pMGA recombinant plasmid was expressed and confirmed by SDS-PAGE and immunoblotting. PvpA-pMGA recombinant protein (20µg and 50µg), ts-11 vaccine strain, and S6 strain that formulated by montanide adjuvant and two control groups (PBS and adjuvant) were injected subcutaneously to six groups of chickens. RESULTS: High yield of protein was purified amount 138 mg/L by affinity batch formation method. Indirect ELISA showed the levels of antibodies in rPvpA-pMGA was significantly higher than ts-11 and S6 groups (p<0.05). The results indicated that antigen-specific response was successfully elicited by the rpMGA-PvpA in chickens. The result of the ELISA with sera collected from ts-11 and S6 groups showed that indirect PvpA-pMGA-ELISA is appropriate candidate for detection of specific antibodies against M. gallisepticum with 100% sensitivity and specificity. CONCLUSIONS: The rPvpA-pMGA is a highly candidate immunogenic protein which induced high amount of humoral immune response. Novel rPvpA-pMGA protein could be useful for evaluation of antibody level in vaccinated poultry flocks.

Role of ESAT-6 in pathogenicity of Beijing and non-Beijing Mycobacterium tuberculosis isolates.

BACKGROUND: Mycobacterium tuberculosis Beijing genotype was associated with tuberculosis outbreaks and increased transmissibility. To understand the variation in virulence between Beijing and non-Beijing clinical isolates of M.tuberculosis genotypes, the esat-6 gene sequencing, and its expression was compared in the macrophage environment. MATERIALS & METHODS: Among 64 nonrepetitive, culture-positive M.tuberculosis, DNA extraction of 24 and 40 pure confirmed Beijing and non-Beijing isolates was accompanied by the boiling method. esat-6 gene PCR amplification and their sequencing were carried out by specific primers and its expression was performed on human macrophage cell line U937 after 6, 12, and 18 h of exposure to bacilli. The esat-6 mRNA transcription and expression in M. tuberculosis treated macrophage by Real-Time PCR and Western blot method. RESULTS: Data analysis based on sequencing of the east-6 gene PCR product showed that this gene exists in all isolates and there are no changes or single nucleotide variation between the Beijing and non-Beijing isolates. In Beijing strains, the esat-6 expression was increased during the study times, but it was constant in non-Beijing isolates. esat-6 gene expression in Beijing isolates reached to about 44.9 times more than non-Beijing isolates after 18 h incubation on the macrophages cell line. CONCLUSION: esat-6 is a conserved gene both in Beijing and non-Beijing isolates of M.tuberculosis. More expression of the east-6 gene in the macrophage model may indicate that this gene is likely to play a more important role in increasing the pathogenicity of Beijing strains.

Crocin Promotes Apoptosis in Human EBV-Transformed B-Lymphocyte via Intrinsic Pathway.

BACKGROUND: As a major carotenoid in saffron, crocin demonstrates potent anti-cancer impacts. However, its anti-lymphoma effects remain vague, especially in the human EBV-associated B-cell lymphoproliferative disorders. This study examined crocin's apoptogenic potential and its underlying mechanism in CO 88BV59-1 cell line vs. normal human peripheral blood B cells. METHODS: CO 88BV59-1 cells were treated with crocin alone or in combination with vincristine for up to 72 h. The cell viability was examined using a resazurin assay. Flow cytometry using annexin V and propidium iodide labeling was performed to detect apoptotic cells. Also, the expression levels of genes and proteins involved in apoptosis (CASP3, CASP8, CASP9, P53, Bax, and Bcl-2) were respectively determined via real-time PCR and Western blot analysis. RESULTS: Crocin concentration-dependently reduced cell viability in CO 88BV59-1 cells with no significant toxicity toward normal B cells. Similar to vincristine, crocin significantly increased apoptosis in these cells during 72 h of incubation. Furthermore, the combination of crocin (80 µM) and vincristine (1 µM) enhanced apoptosis in CO 88BV59-1 cells. Therefore, this synergistic effect was detected in human EBV-transformed B-lymphocyte. CASP3, CASP9, P53, and Bax/Bcl-2 ratio expressions were significantly raised in CO 88BV59-1 cells, whereas CASP8 was unaltered. It was proposed that crocin promoted apoptosis in CO 88BV59-1 cells in a time- and concentration-dependent manner via the induction of the intrinsic pathway. CONCLUSION: The results suggest that crocin may serve as a good alternative/coadjuvant to vincristine in EBV-associated B-cell lymphoproliferative disorders.

Fusion-expressed CtxB-TcpA-C-CPE improves both systemic and mucosal humoral and T-cell responses against cholera in mice.

BACKGROUND: Development of an effective oral vaccine against Cholera, a life-threatening dehydrating diarrheal disease, proved to be a challenging task. To improve oral subunit vaccine immunogenicity and to prevent the state of oral tolerance, application of mucosal adjuvants might be a promising approach. In the present study, the CtxB-TcpA-C-CPE fusion was constructed in which CtxB and C-CPE were used as mucosal adjuvants and vaccine delivery system, respectively, to induce mucosal immune responses, and to improve the anti-toxin and anti-colonizing immunity against V. cholerae. MATERIALS & METHODS: The fusion construct was synthesized, sub-cloned in pQE30 and expressed in E. coli. The three antigen, making the fusion protein, were also separately expressed in E. coli. The recombinant proteins were purified by affinity chromatography using Ni-NTA agarose. Western blot analysis using anti-His antibody was applied to confirm identity of the purified proteins. BALB/c mice were subcutaneously immunized with CtxB, TcpA, C-CPE and the fusion protein CtxB-TcpA-C-CPE separately. The mice were orally immunized (in 3 boosts) by the same vaccine. Mucosal immune response stimulation was evaluated by measuring the levels of intestinal IgA. Systemic immune response was evaluated by measuring total serum IgG, IgG1, IgG2a, IgG2b subclasses, and also IL-4, IL-5, IL-10 and IFN-γ cytokines in spleen cell culture. RESULTS: The recombinant proteins CtxB, TcpA, C-CPE and the fusion protein CtxB-TcpA-C-CPE were expressed in E. coli and highly purified in a single step of chromatography. BALB/c mice immunized with the fusion protein had highest levels of intestinal IgA, serum IgG and IgG subclasses, compared to each of the three proteins making the fusion. Moreover, stimulated splenocytes of mice immunized with the fusion protein displayed significantly higher amounts of IL-5 and IFN-ɣ cytokines. Th2 dominance of the immune response was more evident in mice receiving the fusion protein. CONCLUSION: Inclusion of CtxB, as the mucosal adjuvant, and C-CPE, as the vaccine delivery system, in the fusion protein CtxB-TcpA-C-CPE significantly enhanced the elicited mucosal and systemic immune responses, compared to TcpA alone. Of note, significant production of intestinal IgA in mice immunized with the fusion protein is presumably capable of neutralizing TcpA, CtxB and C-CPE antigens, preventing V. cholera colonization, and toxic function of CtxB and C-CPE. Challenge infection of the immunized mice is required to evaluate protective potential of the fusion protein against V. cholera.

Designing of a chimeric protein contains StxB, intimin and EscC against toxicity and adherence of enterohemorrhagic Escherichia coli O157:H7 and evaluation of serum antibody titers against it.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain is known as one of the major human foodborne pathogens. Lack of effective clinical treatment for human diarrheal diseases confirms the need for vaccine production against enteric bacteria such as E.coli O157:H7. Shiga-like toxin (Stx), EscC, and Intimin are the main important virulent factors of this enteric pathogen. In the present study, a comparative Omics analysis was conducted to identify most invasion EHEC antigenic factors as a potential immunogen. SEI (Stx-EscC-Intimin) trivalent chimeric protein was designed from the exposed and epitope rich part of these virulence factors. Sequence optimization, physicochemical properties, mRNA folding, three-dimensional structure and immunoinformatics data were investigated. The chimeric gene was synthesized with codon bias of E. coli. Recombinant protein was expressed and confirmed by western blot analysis. To evaluate the immunogenicity of the designed protein, the protein was administered to BALB/c mice and the serum IgG was determined by ELISA. Based on the Ramachandran plot, the validation data showed that 90.1 % of residues lie in the favored region. The high antigenicity of the multimeric protein was predicted by the immunoinformatic analysis. Epitope prediction had shown the proper distribution of linear and conformational B-cell epitopes and the competition of T-cell epitopes to bind MHC molecules too. Recombinant ESI Protein with 74.5 kDa was expressed in E. coli. Western blot analysis by anti-Stx antibody, confirmed a single band of chimeric protein. Consequently, the chimeric gene was designed and constructed after assessments. From in silico approach, the protein deduced from this cassette can be an immunogen candidate, and act against toxicity and adherence of EHEC.

Detection and evaluation of rotavirus surveillance methods as viral indicator in the aquatic environments.

Group A rotaviruses (RVAs) have been introduced as the most important causative agents of acute gastroenteritis in the young children. One of every 260 children born globally will die due to rotavirus (RV) before 5 years old. The RV is widely known as a viral indicator for health (fecal contamination) because this pathogen has a high treatment resistance nature, which has been listed as a relevant waterborne pathogen by the World Health Organization (WHO). Therefore, monitoring of environmental is important, and RV is one of the best-known indicators for monitoring. It has been proved that common standards for microbiological water quality do not guarantee the absence of viruses. On the other hand, in order to recover and determine RV quantity within water, standard methods are scarce. Therefore, dependable prediction of RV quantities in water sample is crucial to be able to improve supervision efficiency of the treatment procedure, precise quantitative evaluation of the microbial risks as well as microbiological water safety. Hence, this study aimed to introduce approaches to detecting and controlling RV in environmental waters, and discussed the challenges faced to enable a clear perception on the ubiquity of the RV within different types of water across the world.

Downregulation of hepatitis C virus replication by miR-196a using lentiviral vectors.

Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis and hepatocellular carcinoma. Cellular microRNAs (miRNAs) directly modulate the viral infectivity and indirectly through targeting virus-related host factors. They play an essential role in the progression of different stages of HCV infection. The roles of miR-196 family in HCV infection and hepatocellular carcinoma progression remain poorly understood. Using ViTa databases, miR-196a as a high-score miRNA targeting the NS5 A region of HCV genome was selected. Using dual luciferase assay and an established cell-cultured HCV (HCVcc) system, the effect of miR-196a on HCV genome was assessed. In silico analysis demonstrated the significant role of miR-196a in the downregulation of HCV replication. Using dual luciferase assay, the liver-specific miR-196a and NS5 A gene binding was confirmed. To assess the experimental role of miR-196a, an HCVcc system was established in the Huh 7.5 cell lines. The HCV-RNA 1b derived from an infected patient was transfected into Huh 7.5 cells containing miR-196a lentiviral vectors (Huh 7.5/miR-196a), mocks (Huh 7.5/mock vector), and naïve Huh 7.5 cells. The rate of reduction of the HCV genome replication was assessed using relative real-time PCR assay. These results represent miR-196a overexpression and its roles in regulating HCV genome replication. However, miR-196a may inhibit HCV replication and accelerate the early stages of apoptosis. Overexpression of miR-196a in Huh 7.5 replicon cell is a potential new strategy to prevent hepatitis C infection. The results of this study suggest that miR-196a directly downregulates HCV replication and may serve as a new antiviral therapy.

Production of recombinant lethal factor of Bacillus anthracis in Bacillus subtilis.

Cancer is considered as a disease with high rates of mortality and morbidity. The limitations and side effects of common treatments have prompted the need for innovative cancer therapies. Furthermore, selectivity and targeting of cancer cells are crucial factors to successful treatment of cancer. One of these methods is the use of bacterial toxins including Bacillus anthracis toxin to aid cancer therapy. This toxin is composed of three polypeptides: protective factor (PA), lethal factor (LF), and edema factor (EF). PA can bind to various surface receptors of all types of human cells and it internalizes the lethal factor and edema factor subunits of the toxin in the cytosol. In the present study, we cloned and expressed the lef gene of B. anthracis as the lethal part of the toxin in Bacillus subtilis WB600 by a shuttle expression vector PHT4. The rLF made in B. subtilis is efficiently secreted by the host into the culture medium which facilitates downstream processing. The rLF can be used to study cancer treatment. Abbreviations: EF: edema factor; LF: lethal factor; PA: protective factor; rLF: recombinant lethal factor; rPAm: recombinant protective factor mutants; uPA: urokinase-type plasminogen activator; uPAR: urokinase-type plasminogen activator receptor.

Molecular Monitoring and Phylogenetic Analysis of Betanodavirus in Groupers (Epinephelus spp.) and Asian Sea bass (Lates calcarifer) of Iranian Northern Waters of the Persian Gulf.

The emergence of diseases has caused much health and economic damage. Viral Nervous Necrosis (VNN) is considered as one of the most important threats to aquatic ecosystems. VNN can cause severe mortality and economic loss in fish farms. The high water temperatures in southern Iran and the observed incidences of fish mortality in the Persian Gulf led to the hypothesis of the possible emergence of VNN. Therefore, this study aimed to monitor two species of fish susceptible to VNN using PCR, and Nested PCR methods and comparing the sensitivity of these methods to the identification of Betanodavirus infection in apparently healthy and symptomatic fish. About 850 Grouper (Epinephelus spp.) and Asian Sea bass (Lates calcarifer) fish of the Persian Gulf were collected randomly and examined. Molecular methods were used to identify NNV in visibly healthy and symptomatic fish of the Persian Gulf of Iran. The results of the PCR showed no positive cases, but the Nested PCR revealed some positive results. Then, the phylogenetic analysis of the virus sequence was performed. The nucleotide sequence of Nested PCR products revealed a 98-100% homology with Red Spotted Grouper Viral Nervous Necrosis (RGNNV). This is the first report on VNN tracing and detection as well as phylogenetic analysis of the virus from the Persian Gulf of Iran. Therefore, considering the importance of emerging viral diseases and the irreparable damage they cause, continuous monitoring and epidemiological studies of VNN were recommended by authorized organizations.

Comparison of two molecular diagnostic methods for identifying Beijing genotype of Mycobacterium tuberculosis.

BACKGROUND AND OBJECTIVES: The Beijing family of Mycobacterium tuberculosis has been identified as a severe pathogen among this species and found in many clinical isolates during the last decade. Early identification of such genotype is important for better prevention and treatment of tuberculosis. The present study performed to compare the efficiency of Real-Time PCR and IS6110-Based Inverse PCR methods to identify the Beijing family. MATERIALS AND METHODS: This study was carried out on 173 clinical isolates of Mycobacterium tuberculosis complex in Golestan Province, northern Iran. DNA extraction performed by boiling and determining the Beijing and non-Beijing strains carried out using Real-Time PCR and IS6110-Based Inverse PCR. RESULTS: In both Real-Time PCR and IS6110-Based Inverse PCR method, 24 specimens (13.9%) of the Beijing family were identified and the result of the IS6110-Based Inverse PCR method showed that all the Beijing strains in this region belonged to the Ancient Beijing sub-lineage. CONCLUSION: Although the efficacy of the two methods in the diagnosis of the Beijing family is similar, the IS6110-Based Inverse PCR is more applicable to the ability to detect new and old Beijing family.

A synergistic association between adhesion-related genes and multidrug resistance patterns of Staphylococcus aureus isolates from different patients and healthy individuals.

OBJECTIVES: Biofilm -forming capacity of Staphylococcus aureus (S. aureus) as a commensal opportunistic bacterial species induce a growth in antibiotic resistance in chronic diseases. Since expression of biofilm- related genes and antibiotic resistance function are interdependent, the present study was an attempt to inquire biofilm formation and its relationship with antibiotic resistance in clinical isolates. METHODS: 208 S. aureus clinical isolates from four major provinces of Iran were investigated in terms of presence of adhesion genes (icaA, icaD, icaB, icaC, fnbpA, fnbpB, clfA, clfB, cna, sasC, sasG and bap) using PCR. In addition, microtiter plate (Mtp) assay was performed to examine quantitative biofilm formation of the isolates and their antibiotic resistance patterns against 16 antibiotics determined upon CLSI criteria. RESULTS: The results revealed high prevalence rate (almost 100%) of icaADBC and MSCRAMMs genes in the isolates. Moreover, bap gene was not detected in any of the tested clinical isolates. Based on phenotypic method 169 isolates (81.25%) were also found to have biofilm formation ability. Among 208 isolates, 98 (47.12%) isolates were multidrug resistant (MDR). Vancomycin, linezolid, nitrofurantoin and quinupristin/dalfopristin were the most effective drugs against MDR strains. Furthermore, the findings demonstrated a significant relationship between MDR and biofilm forming capacity. CONCLUSION: Prevalence rate of adhesion- related genes was high in S. aureus from isolates in Iran ;so these genes might be expressed under certain conditions and cause emergence of MDR strains. Therefore, further investigations are necessary to prevent initial attachment based on new candidate adhesion genes for vaccine design.

The impact of Helicobacter pylori infection on gut microbiota-endocrine system axis; modulation of metabolic hormone levels and energy homeostasis.

The gut microbiota is a complex ecosystem that is involved in the development and preservation of the immune system, energy homeostasis and nutritional status of the host. The crosstalk between gut microbiota and the host cells modulates host physiology and metabolism through different mechanisms. Helicobacter pylori (H. pylori) is known to reside in the gastric mucosa, induce inflammation, and alter both gastric and intestinal microbiota resulting in a broad spectrum of diseases, in particular metabolic syndrome-related disorders. Infection with H. pylori have been shown to affect production level and physiological regulation of the gut metabolic hormones such as ghrelin and leptin which are involved in food intake, energy expenditure and body mass. In this study, we reviewed and discussed data from the literature and follow-up investigations that links H. pylori infection to alterations of the gut microbiota and metabolic hormone levels, which can exert broad influences on host metabolism, energy homeostasis, behavior, appetite, growth, reproduction and immunity. Also, we discussed the strong potential of fecal microbiota transplantation (FMT) as an innovative and promising investigational treatment option for homeostasis of metabolic hormone levels to overcome H. pylori-associated metabolic syndrome-related disorders.

Molecular detection and genotyping of group A rotavirus in two wastewater treatment plants, Iran.

In different countries especially developing countries, treatment of urban wastewater might be ineffective removal of pathogens such as group A rotavirus. The objective of this study is to evaluate the efficiency of rotavirus removal in two wastewater treatment plants (WWTPs) in Isfahan, Iran. To meet the study objectives, 96 sewage samples from influent (n = 48) and final effluents (n = 48) were collected by grab sampling. Two different concentration methods included pellet and two-phase used for concentrating sewage samples. The presence of rotavirus antigens in all concentrated sewage samples was screened by enzyme-linked immunosorbent method. To analyze the study samples, real-time PCR technique with SYBR Green I fluorescent dye and nested multiplex PCR for rotavirus genotyping were utilized respectively. Result indicated positive rotavirus percentage in two methods of ELISA and real-time PCR was equal to 61.45% (59 cases) and 44.79% (43 cases). In addition, analyzing seasonal distribution of rotavirus shows different distributions as below: in spring (18.64%), summer (20.33%), autumn (35.60%), and winter (25.42%). Finally, rotaviruses illustrate significantly higher frequency in cold seasons. G10 and G1 types are considered the most, among common genotypes which identified in 11 (25.58%) and 5 (11.62%), out of the 43 positive samples in WWTPs, followed by non-typeable genotypes (13.95%) and mix genotypes (11.62%); and different genotypes including G2, G3, G4, G8, G9, and G12 were equal to 2.33, 9.30, 9.30, 2.33, 7, and 7% in the WWTPs, respectively. Such high prevalence underlines the significance of environmental surveillance. Also, to eliminate potential pathogens especially enteric viruses from sewage, the improvement of treatment systems is essential.

Molecular surveillance of human rotaviruses in drinking water and investigation of the efficiency of their removal in Isfahan water treatment plant.

Enteric viruses, especially human rotaviruses present in aquatic environments, are microbial criteria in quality assessment of water resources. The present research aimed to investigate molecular monitoring of human rotavirus and efficacy evaluation of Isfahan water treatment plant (WTP) in the elimination of viruses. In total, 60 water samples were collected from different units of WTP. Zeta plus electropositive Virosorb cartridge filter and elution buffer was used for concentrating water samples. Enzyme-linked immunosorbent assay (ELISA) was used for detecting rotavirus antigen. Quantitative real-time reverse transcription PCR (qRT-PCR) with SYBR Green I fluorescent dye was performed for molecular detection of rotavirus. Multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) was used for rotavirus G genotyping. Total coliform count varies from 102-103 CFU/mL in the raw water resources. Rotavirus antigen was detected in 17 samples (28.33%) by ELISA, and 13 samples (21.67%) were found positive by RT-PCR. These included 41.18% (7 cases) of raw water influent, 29.41% (5 cases) after sedimentation, 23.52% (4 cases) after ozonation, and 5.88% (1 case) after filtration in ELISA method. The highest number of rotaviruses was detected by qRT-PCR in autumn (46.15% (6 cases)). The commonest circulating G type in the sampling points was the mixed types, which was identified in 6 samples (46.15%), followed by non-typeable (23.07%), G3 (15.38%), G1 (7.69%), and G8 (7.69%), respectively. Despite the presence of rotavirus in raw water, after clarification and ozonation, filtration and treated water did not show the presence of rotavirus. The results of this study showed that multi-stage treatment has a positive effect on virus removal in WTP.

Study of the immunogenicity of outer membrane protein A (ompA) gene from Acinetobacter baumannii as DNA vaccine candidate in vivo.

OBJECTIVES: Acinetobacter baumannii is one the most dangerous opportunistic pathogens in hospitalized infections. This bacterium is resistant to 90% of commercial antibiotics. Therefore, developing new strategies to cure A. baumannii-infections is urgent. The DNA vaccines new approach in which the immunogen can be directly expressed inside the target cells through cloning of immunogen into an expression vector. The outer membrane protein A(OmpA) is one the critical factors in pathogenicity of A. baumannii which has been repeatedly described as a powerful immunogen to trigger the immune responses. As the pure form of the OmpA is insoluble, vaccine delivery is very hard. MATERIALS AND METHODS: We previously cloned the ompA gene from A. baumannii into the eukaryotic expression vector pBudCE4.1 and observed that the OmpA protein has been considerably expressed in eukaryotic cell model. In current study, the immunogenic potential of pBudCE4.1-ompA has been evaluated in mice model of experimental. The serum levels of IgM, IgG, IL-2, IL-4, IL-12 and INF-γ were measured by enzyme-linked immunosorbent assay (ELISA) after immunization with ompA-vaccine. The protective efficiency of the designed-DNA vaccine was evaluated following intranasal administration of mice with toxic dose of A. baumannii. RESULTS: Obtained data showed the elevated levels of IgM, IgG, IL-2, IL-4, IL-12 and INF-γ in serum following the vaccine administration and mice who immunized with recombinant vector were survived more than control group. CONCLUSION: These findings indicate ompA-DNA vaccine is potent to trigger humoral and cellular immunity responses although further experiments are needed.

Cloning and expression of a novel synthetic gene containing VP1 and 3A in Bacillus subtilis as a vaccine candidate against foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock animals and control of the disease based on vaccination against serotypes O, A and Asia 1 is important. VP1 (structural) protein and 3A (non-structural) protein is the important antigen in FMDV and they can be used to design recombinant vaccines. In this study the bioinformatics characteristics of VP1 [141-160 and 23-42] and 3A [21-35] of Iranian serotypes O, A and Asia 1 was obtained using on-line predicting software. Then the sequence VP1 [141-160]-GS-VP1 [23-42]-GS-3A [21-35]-GS were codon-optimized and cloned onpHT43shuttle vector and finally expressed in Bacillus subtilis WB600 strain. We could predict VP1 [141-160] as a B cell epitope, VP1 [23-42] as a CTL epitope and 3A [21-35] as a Th cell epitope. The 20KD recombinant protein expressed by Bacillus subtilis were detected by SDS-PAGE. The results showed that this recombinant protein had epitope characteristics and it could be useful as a vaccine candidate to control all serotypes of FMD in Iran.

La amplia distribución de un microorganismo termoacidófilo en el mineral de una mina cuprífera a temperatura ambiente y en condición ácida, y su importancia en la biolixiviación del concentrado de calcopirita / The wide distribution of an extremely thermoacidophilic microorganism in the copper mine at ambient temperature and under acidic condition and its significance in bioleaching of a chalcopyrite concentrate

Thermoacidophiles can exist in a state of dormancy both in moderate temperatures and even in cold conditions in heap leaching. Sulphide mineral ores such as chalcopyrite produce sulfuric acid when exposed to the air and water. The produced sulfuric acid leads to the decrease of pH and exothermic reactions in heap leaching causing the temperature to increase up to 55 °C and the activation of thermoacidophilic microorganisms. The aim of the present study was to isolate indigenous extreme thermoacidophilic microorganisms at ambient temperature from Sarcheshmeh Copper Complex, to adapt them to the high pulp density of a chalcopyrite concentrate, and to determine their efficiency in chalcopyrite bioleaching in order to recover copper. In this study samples were collected at ambient temperature from Sarcheshmeh Copper Complex in Iran. Mixed samples were inoculated into the culture medium for enrichment of the microorganisms. Pure cultures from these enrichments were obtained by subculture of liquid culture to solid media. Morphological observation was performed under the scanning electron microscope. Isolates were adapted to 30% (w/v) pulp density. For the bioleaching test, the experiments were designed with DX7 software. Bioleaching experiments were carried out in Erlenmeyer flasks and a stirred tank reactor. The highest copper recovery in Erlenmeyer flasks was 39.46% with pulp 15%, inoculums 20%, size particle 90 pm and 160 rpm. The lowest recovery was 3.81% with pulp 20%, inoculums 20%, size particle 40 pm and 140 rpm after 28 days. In the reactor, copper recovery was 32.38%. Bioleaching residues were analyzed by the X-ray diffraction (XRD) method. The results showed no jarosite (KFe3(SO4)2(OH)6) had formed in the bioleaching experiments. It seems that the antagonistic reactions among various species and a great number of planktonic cells in Erlenmeyer flasks and the stirred tank reactor are the reasons for the low recovery of copper in our study.

Los microorganismos termoacidófilos pueden estar en estado latente tanto a temperatura moderada como baja, en lixiviación en pilas. Los minerales sulfurosos, como la calcopirita, producen ácido sulfúrico cuando se exponen al aire y al agua. El ácido sulfúrico producido conduce a la disminución del pH y a reacciones exotérmicas durante la lixiviación en pilas, lo que hace que la temperatura aumente hasta 55 °C y se activen los microorganismos termoacidófilos. El objetivo del presente estudio fue aislar del complejo de cobre Sarchesh-meh (Irán) microorganismos termoacidófilos extremos que proliferan a temperatura ambiente e investigar su adaptación a la alta densidad de pulpa del concentrado de calcopirita, así como su eficiencia para biolixiviarese mineral, con el objeto de recuperar el cobre. Se recogieron muestras a temperatura ambiente del citado complejo, y luego muestras mixtas se inocularon en un medio de cultivo de enriquecimiento. A partir de estos enriquecimientos, mediante el subcultivo del cultivo líquido a medio sólido, se obtuvieron cultivos puros. La observación morfológica se realizó bajo microscopio electrónico de barrido. Los aislados estaban adaptados al 30% p/v de densidad de pulpa. Para la prueba de biolixiviación, los experimentos fueron diseñados con el software DX7. Los experimentos de biolixiviación se llevaron a cabo en Erlenmeyers y en un reactor tanque con agitación. La mayor recuperación de cobre en los Erlenmeyers fue del 39,46% y se obtuvo con la pulpa al 15%, un inóculo del 20%, un tamaño de partícula de 90 µm y una agitación de 160 rpm. La menor recuperación fue del 3,81% y se obtuvo con la pulpa al 20%, un inóculo del 20%, un tamaño de partícula de 40 µm y una agitación de 140 rpm, después 28 días. En el reactor, la recuperación del cobre fue del 32,38%. El análisis de difracción de rayos X (XRD) no mostró que se formara jarosita (KFe3-#91;SO4-#93;2-#91;OH-#93;6) en los experimentos de biolixiviación. Dicha técnica sirve para determinar la estructura cristalina de una sustancia desconocida. Al parecer, las reacciones antagónicas entre las diversas especies y el mayor número de células planctónicas en los Erlenmeyers y en el reactor fueron las causas de la baja recuperación de cobre observada en este estudio.

The wide distribution of an extremely thermoacidophilic microorganism in the copper mine at ambient temperature and under acidic condition and its significance in bioleaching of a chalcopyrite concentrate.

Thermoacidophiles can exist in a state of dormancy both in moderate temperatures and even in cold conditions in heap leaching. Sulphide mineral ores such as chalcopyrite produce sulfuric acid when exposed to the air and water. The produced sulfuric acid leads to the decrease of pH and exothermic reactions in heap leaching causing the temperature to increase up to 55°C and the activation of thermoacidophilic microorganisms. The aim of the present study was to isolate indigenous extreme thermoacidophilic microorganisms at ambient temperature from Sarcheshmeh Copper Complex, to adapt them to the high pulp density of a chalcopyrite concentrate, and to determine their efficiency in chalcopyrite bioleaching in order to recover copper. In this study samples were collected at ambient temperature from Sarcheshmeh Copper Complex in Iran. Mixed samples were inoculated into the culture medium for enrichment of the microorganisms. Pure cultures from these enrichments were obtained by subculture of liquid culture to solid media. Morphological observation was performed under the scanning electron microscope. Isolates were adapted to 30% (w/v) pulp density. For the bioleaching test, the experiments were designed with DX7 software. Bioleaching experiments were carried out in Erlenmeyer flasks and a stirred tank reactor. The highest copper recovery in Erlenmeyer flasks was 39.46% with pulp 15%, inoculums 20%, size particle 90µm and 160rpm. The lowest recovery was 3.81% with pulp 20%, inoculums 20%, size particle 40µm and 140rpm after 28 days. In the reactor, copper recovery was 32.38%. Bioleaching residues were analyzed by the X-ray diffraction (XRD) method. The results showed no jarosite (KFe3(SO4)2(OH)6) had formed in the bioleaching experiments. It seems that the antagonistic reactions among various species and a great number of planktonic cells in Erlenmeyer flasks and the stirred tank reactor are the reasons for the low recovery of copper in our study.