The microbiological quality of drinking water harbored serious importance for public health. To assess the efficiency of household water membrane systems for eliminate the bacteria and due to high clinical relevance of Pseudomonas aeruginosa, presence and characterization of this bacterium in the filters of plant were investigated. To this aim filters of 50 household water membrane systems were studied.
Microbiological and molecular methods were performed for detection and conformation of P. aeruginosa isolates. The antimicrobial susceptibilities of isolates were determined using the standard Kirby–Bauer disc diffusion method. The antibiotics used included: piperacillin, ticarcillin, ciprofloxacin, ceftazidime, cephepime, and imipenem. Combined disk (CD) method and double disk synergy test (DDST) were performed for metallo-beta-lactamase production in P. aeruginosa isolates. Finally, PCR was performed to detect MBL genes, in MBL-producing strains.
Of the 50 plants, which were analyzed, 100 colony were identified as P. aeruginosa. In some plants, isolated bacteria from different filters harbored similar or close genetic profile. This means that these isolates were likely able to pass through the filter and reach higher filters of the device. The phenotypic tests revealed MBL production in 7 (7%) strains. Tow isolates were positive for blaVIM-1 while one isolate showed blaNDM and blaIMP-1. The highest level of resistance was against to ticarcillin 30 (30%),. One P. aeruginosa isolate was resistant to the entire antimicrobial disc test. Ten isolates (10%) were resistant to at least three antibiotic classes.
Wide distribution of resistant phenotypes and genetic plasticity of these bacteria in household water membrane systems may indicate that resistance mechanisms circulate in the environment of systems between environmental P. aeruginosa isolates. The presence of MBL-producing genes in these environments and P. aeruginosa as a potential reservoir of these resistance genes is leading to concern for public health.

Empiric therapy for staphylococcal infections by penicillins led to spread of penicillin resistance throughout the world. Therefore reliance on vancomycin for treatment of documented methicillin-resistant S. aureus (MRSA) infections, as well as for empiric therapy of infections in populations where the prevalence of MRSA is high is increased. Since 2002, nine MRSA strains that are also resistant to vancomycin (VRSA) have been reported in the United States. There is no documented evidence for investigation of vancomycin resistance among Staphylococcus isolates in Khouzestan. A total of 100 MRSA isolates collected from clinical specimens of patients in hospitals of Khuzestan province and checked for presence of vancomycin resistance genes (vanA, vanB, vanC, and vanD) by PCR. Also phenotypic resistance of isolate was checked by minimum inhibition concentration (MIC) method. Among the isolates prevalence of vanA, vanB, vanC, and vanD was found 1%, 0%, 1% and 1% respectively. one isolate showed MIC equal to 16 µg/ml, one isolate showed MIC equal to 8 µg/ml, one isolate showed MIC equal to 4 µg/ml. The results showed that the frequency of vancomycin resistance genes is high in MRSA strains isolated from patients in south of Iran. Thus to keep the emergence and transmission of these isolates to a minimum, urgent interventions are needed

 Staphylococcus aureus resistance to methicillin (MRSA) is one of important worldwide health problem. The Gram – positive pathogen Staphylococcus aureus is the most important human pathogen causing skin and tissue infection. To counteract innate immunity S. aureus expresses balanced immune proteins in an immune evastion cluster (IEC) called: staphylokinas, staphylococcal enterotoxin– p, staphylococcal enterotoxin – A, staphylococcal complement inhibitor, chemotaxis inhibitory protein. Genes are located on bacteriophages. The IEC-carrying bacteriophages are called β-haemolysin-converting bacteriophages (ΒC-ØS). It was shown that 90% of S.aureus strains contain an IEC- carrying ΒC-Øs. This study was conducted to determine the prevalence rate, of sea, sep, chp, sak and scn genes, antibiotic pattern and typing these genes in Staphylococcus aureus isolated from patiens in hospitals of Ahvas. In this descriptive study, 155 samples were isolated from hospital patiens during 9 month. Evalution and assessment of antibiotic resistance showed the greatest resistant to penicillin (97%), methicillin (94%), cefoxitin (93.5), ceftazidime (77.5), erythromycin (72), clindamycin (62%), ciprofeloxacin (60%), gentamycin (54%), and imipenem (55%) respectively. Polymerase chain reaction (PCR) method was used to estimate the prevalence of these genes. Out of the 155 isolates, 94 (61%), 44 (28%), 60 (39%), 54 (35%), 5 (3.2 %) were reported positive to sak, chp, scn, sea, and sep genes respectively. Typing of immune evasive cluster (IEC) showed type A (5.1%), type B (10.5%), type C (13.5%), type D (12.25%), type E (28%), type F (1.3%), type G (0.65%). Howewer, 15.5% of isolates showed any of IEC types. In conclusion, the large variety observed in IECs expressed that this gene cluster is very dynamic. This provides for S. aureus a unique mechanism to adapt and counteract with human body immune system. The undrastanding of IEC role in virulence will enhance our knowladge about evolutionary potentials of S. aureus.

Bovine viral diarrhea virus (BVDV) is a common pathogen of cattle herds that causes economic losses due to reproductive disorders in breeding cattle. The development of rapid and reliable diagnostic methods is critical for disease diagnosis and control program.
In this study, the specific agglomerative behavior of peptide nucleic acids (PNA) with gold nanoparticles was manipulated by its complementation with viral RNA to design a rapid visual label-free assay for viral RNA detection of BVDV. To design the PNA, 5′ Untranslated (5′UTR) sequences of BVDV-1 and BVDV-2 were retrieved from GeneBank; aligned and conserved part was selected. Reaction was designed based on different concentrations of PNA, AuNPs and RNA. Charge neutral PNA are used as a “coagulant” of citrate anion-coated particles and as hybridization probe. In the absence of a complementary target RNA, free PNA molecules in solution induce aggressive particle aggregation because of the removal of charge repulsion as a result of PNA coating on nanoparticles. When a complementary DNA is present and PNA-RNA complexes are formed, the particles remain stable because the negative charges of the DNA strands in the complexes adsorbed on the particle surface ensure sufficient charge repulsions. The sensitivity and specificity of the projected assay was compared to existing tests, including Real-Time RT-PCR and RT-nested multiplex-PCR tests.
The colorimetric changes showed apparent color stability at 10.48 ng/μl of RNA, which was indicative of the visual detection limit for the viral RNA, whereas spectral changes for the corresponding solutions helped determine the presence of viral RNA at concentrations of 1.05 ng/μl RNA, providing a detection limit for the given assay using a spectrophotometer. The detection limit of Real-Time RT-PCR and RT-nested multiplex-PCR tests was found to be 10.48× 10-5 ng/μl and 41.92× 10-4 ng/μl respectively. The PNA-induced colorimetric changes in the presence of the target virus at different RNA concentrations with a linear coefficient of R2 = 0.992, which shows linear correlation for quantitative assay.
Although Real-Time RT-PCR and RT-nested multiplex-PCR assay have higher sensitivity, the present assay was much faster, simple and cost effective; moreover have acceptable sensitivity and specificity and is suitable option when need quick diagnosis.
 

The water is one of the first needs of human life and the most effective factors in the material and force cycle in nature that have vital role for human and civilization life. The quality and safety of the drinking water still is an important public health issue. In recent years, due to pollution of drinking water, membrane technologies have been established. The household filter systems reduce the pollutions that flavor, smell, turbidity and unfavorable effects cause. But the quality of filters is decreased by formation of biofouling. Biofouling is growth of microorganisms and produce microbial biofilm. In bacteria, the transfer of the antibiotic resistance genes is probability produce by bifilm formation. According to the possibility of microbial contamination of the memebranes, this project was performed to investigate the membrane of household filter systems in Ahvaz city and finally identification of antibiotic resistance in these isolates.
In these work, 50 household filter systems membranes was studied. Certain amount of each filter was taken and cultured in nutrient broth. Total colony of each filter was counted. The bacteria were identified by differential and biochemical tests. The antibiotic resistance was carried out by disc diffusion method.
Colony count showed that different ranging from 105 to 109 of bacteria can growth on the surface of filters. These bacteria was identified as Pseudomonas, Bacillus, Rhodococcus, Achromobacter and Sphingomonas. Antibiogram tests for them showed that most of them are multi-drag resistance (MDR) bacteria. The Random Amplified Polymorphic DNA (RAPD) analysis showd that P.aueroginosa isolated have different genotype patterns while some of them that isolated from different membranes have a similar patterns.
The results show that household filter systems membranes can be a suitable environment for producing of biofilm. MDR bacteria can transfer antibiotic resistance genes to sensitive bacteria or body microflora via drinking water. Pseudomonas genus was dominant and was isolated from most of the household filter systems.
 

Foot-and-mouth disease (FMD) is a highly contagious and acute disease that is caused by FMD virus (FMDV) in domestic and wilds cloven-hoofed animals.The importance of FMD is due to huge economic losses in the livestock production. In Iran, this disease is the most threatening factor of livestocks, and is on the top of the list of animals disease control program. Alhagi maurorum Medik. is a shrub from Papilionaceae family that is used widely in traditional medicine. According to it’s tissue healing and anti- bacterial properties, this study was done in order to evaluate its ethanloic, methanolic and aqueous-acetic acid extract’s antiviral activity against FMDV, via Baby Hamster Kidney (BHK) cell culture. The plant was collected and extracts were prepared; then non-cytotoxic concentration of extracts were evaluated by MTT-assay and 50% cytotoxicity concentration (CC50) was measured. Then serial dilutions of non-toxic concentration of extracts were prepared and their antiviral activity was examined by colorimetric MTT- assay and plaque reduction assay, in different virus activity phases . The 50% inhibitory concentration (IC50) was calculated and selectivity index (SI) was declared by CC50/IC50. Results revealed that ethanolic and aqueous extracts were non- cytotoxic in 0.3 mg/ml or less and methanolic extract was non-cytotoxic in concentration of 3 mg/ml or less. The extracts had prominent inhibitory activity on free virion (virucidal assay) and pre-treatment assay. Otherwise the less inhibitory activity was observed in co-treatment assay and post-treatment assay.Ethanolic extract has more significant antiviral activity and methanolic extract has the least cytotoxic activity. The selectivity index ranged from 3.19 to 135.50.

Anelloviruses have a single-stranded, circular, negative-sense DNA genome and classified as members of the new family Anelloviridae.This family includes nine genera (Alphatorquevirus to Zetatorquevirus) that can infect humans and animals. There are afew studies about TTMDV/SAV. The aim of this study was investigation of TTMDV presence in volunteer blood donor in Ahvaz city and their´s relation with hepatitis B virus .Sixty healthy samples (negative for hepatitis B) and 42 hepatitis B positive samples were collected from blood transfusion center in Ahvaz. After DNA extraction from samples, the quality of extracted DNA was investigated and to assay the presence of TTMDV in healthy and Hepatitis B virus infected, nested-PCR was done. Relative frequency of TTMDV in individuals affected by hepatitis B and healthy cases were 11.9% and 13.3%, respectively. This difference was not mean for statistical analysis (P>0.05). Φ-square test showed that there are no relation between Hepatitis B virus and TTMDV (P>0.05). The frequency of TTMDV in male and female person was 13.9% and 10%, respectively. Fischer's exact test showed that this difference was not mean for statistical analysis (P>0.05). Logistic regression showed that infection chance with TTMDV is decreased by age enhancing (Reduction ratio 0.98 and confidence interval 95% and 0.93-1.04)

 hepatitis B virus (HBV), one of the major factors acute and chronic hepatitis that can lead to cirrhosis liver and Hepatocellular Carcinoma.Healthcare personnel and medical students are at a greater risk of getting infectious disease including hepatitis B than other groups of society because of their frequent exposure to patients and blood products and derivatives as well as secretions for different parts of patient's body. One of the prevention ways is immunizing people against these diseases by performing vaccination among community members. The aim of this study was to have an analysis on healthcare personnel immunity and internal cell expression of interferon gamma ( INF- ) stimulated by hepatitis B virus in in vitro condition.This study was performed on temporal basis and has been affected on 130 persons of healthcare and treatment network's personnel in Dasht Azadegan that received recombinant hepatitis B Vaccine. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Density Gradient Centrifugation method and cultured in RPMI1640 at 37 ˚C in a humidified 5% CO2 incubator. To stimulate the cells, 15 of Hepatitis B Virus Antigen (20 / ml), recombinant HBsAg, was added to culture medium containing PBMC (to each well). Supernatant of culture medium was analyzed by ELISA Kit for assessment of INFγ production. Samples were also analyzed for hepatitis B anti-virus antibody level by ELISA method. Results showed that 68.5 % of all personnel had a good immunity response (10 MIU<) and 31.5 % had a response to vaccination (<10 MIU). The amount of INFγ was at which there is a direct relationship between the concentration of INFγ and anti-HBs titration among persons that have been studied in this study (r=0.653). There was a significant relationship between the amount of INFγ and anti-HBs Ab titration with Body Mass Index (BMI), the number of vaccine doses, time interval with the last vaccine injection (P <0.05). the results of this study showed that 30.76% of studied persons had no immunity by receiving hepatitis B vaccine. It is recommended that 5 years after vaccination, level of HBs Ab should be tested in under-threat persons and then have a decision on recalling dose.

Name: Zahra First name: Heidari

Title: Detection of Bovine viral diarrhea virus using gold Nanoparticles

Supervisor: Dr Seyedeh Elham Rezatofighi
Advisors: Dr Sadat rastegarzade
Grade: MSc  Major: Biology  Field: Microbiology
University: Shahid Chamran University of Ahvaz  Faculty: Science
Date of graduate: 1393/ 11 /5 pages:104
Keywords: Bovine viral diarrhea virus, Gold nanoparticles, RT-nested multiplex-PCR
Abstract: Bovine viral diarrhea virus (BVDV) is an agent responsible for cattle infections and has economic importance worldwide. It causes considerable financial losses to the livestock industry. In current study, detection of Bovine Viral Diarrhea was performed via three different methods using gold nanoparticles and reaction conditions were optimized. These methods were unmodified gold nanoparticles, modified gold nanoparticles via one and two probes. In these methods, the color change of gold nanoparticles was observed by armless eyes. Fifthly specimens of BVDV-suspected infectious bloods were collected and tested by both gold nanoparticles and RT-nested multiplex-PCR methods. Of twenty BVDV-positive specimens detected in RT-nested multiplex-PCR 18 specimens in two-probe modified gold nanoparticles, 17 specimens in single-probe modified nanoparticles and 16 specimens in unmodified gold nanoparticles were confirmed to be BVDV-positive. As compared with RT- nested multiplex-PCR, the sensitivity and specificity of modified two-probe gold nanoparticles were 90% and 96.66% but modified single-probe gold nanoparticles showed 85% and 90% sensitivity and specificity and unmodified gold nanoparticles 80% and 83/3% respectively. In comparison, although nanoparticles method had less sensitivity than RT-nested multiplex PCR it was rapid, cost-effective and more simplified method.

 

Bovine viral diarrhea virus (BVDV) is a pathogen that infects cattle, and has worldwide economic importance. It causes considerable financial losses to the livestock industry. In this study, a simple strategy of colorimetric RNA detection is presented based on a hairpin assembly reaction and target-catalytic RNA circuits to achieve enzyme-free amplification. The method employed two hairpin probes (H1 and H2), which were stable and unable to hybridize in the absence of target. In the presence of target, the target was hybridized with hairpin H1 and the opened hairpin H1 hybridized with hairpin H2. Opened hairpin H2 was complementary to H1 and hybridized with H1 and so on. In the absent of the target, single-stranded DNA (ssDNA) sticky ends of probes stabilized AuNPs and effectively prevented them from salt-induced aggregation. The target RNA was hybridized with the hairpin auxiliary probes and triggered the formation of extended double-stranded DNA (dsDNA) polymers through HCR. As a result, the formed dsDNA polymers provide less stabilization without ssDNA sticky ends, and AuNPs undergo aggregation when salt concentration was increased. Subsequently, a pale purple-to-blue color variation is observed in the colloid solution. Fifty blood specimens were collected from bovines suspected to suffer from BVDV infection, and were tested in parallel by HCR and nested multiplex RT-PCR. The HCR detection limit was estimated to be approximately 0.1 TCID50 /μL of virus. Comparison of nested multiplex RT-PCR with HCR in this study revealed that the newly developed HCR based on gold nanoparticle assay is a highly sensitive and specific method for BVD virus detection in clinical samples. The method may have a wide range of sensor applications because it is enzyme-free and simple to perform.

FMD is a highly contagious and acute disease that affects some of domestic and wilds ruminants animal such as cattle, sheep, deer and pigs. Losing livestock would directly affect the economy and hence understanding and controlling FMD is becoming more and moe important. In our country this disease is the most important factor threatening the captal and animal products and is on the top of the list of animals disease control. In this study the antiviral effect of magnesium nanoparticles, silver and gold on FMDV in cell culture were studied. Magnesium nanoparticles were the least toxic to the cells and lower than 250 µg/ml had almost no toxic affect. Moreover lower than 1.56 µg/ml of gold nanoparticles had no toxic effects. Silver nanoparticles were more toxic to the cells so that the concentration of 15.62 ng/ml was without toxic effects. Antiviral effects of nanoparticles on different stages of virus growth was investigated by plaque reduction assay. The magnesium nanoparticles can inhibit the free virion and had no effect on the intracellular virus. Silver nanoparticles have the greatest effect on the virus adsorption step and similar to magnesium nanoparticles it has no impact on the virus inside the cell. Unlike the other two nanoparticles, the gold nanoparticles were able to inhibit virus reproduction. This study shows different affects of the nanoparticles on the virus.