فصلنامه دنیای میکروب ها
سال دوم شماره 4 (پیاپی 5، زمستان 1388)

Core+1 or F protein is recently identified to be encoded by hepatitis C virus (HCV) genome. Although functional properties of this protein are not still discovered, core+1 seems to play important roles in viral life cycle and pathogenesis. This study seeks to clone and express HCV core+1 gene in a eukaryotic system with the final aim of evaluating its potential roles in cell signaling pathways and resistance of HCV to therapy.

Core+1 gene corresponding to the amino acids 1-162 was PCR-amplified from the original gene of subtype b that was previously cloned in a prokaryotic vector (14). The amplicon harboring 6xHis-tag at C-terminal was subsequently subcloned in the eukaryotic pCDNA3.1+ vector. The Nhe I / BamHI restriction sites, initiation and stop codons as well as kozak sequences were designed in the primers. Constructed plasmid was verified by restriction enzyme analysis and sequencing reactions. Liver cell line of Huh7 was transfected with this plasmid using electroporation and lipofection techniques. Transfection efficiency was checked by co-transfection of pEGFPN1 plasmid and flowcytometry. Finally expression and localization of core+1 protein was evaluated by means of confocal microscopic technique.

Restriction enzyme analysis and sequencing reactions indicated the accuracy of cloning procedure. Flow cytometric analysis showed higher electroporation efficiency for transfection of Huh7 cells, in comparison with lipofection method. Finally, results of confocal microscopic microscopy using anti-His antibody confirmed the transcription and expression of core+1 protein in Huh7 cells.

Our study resulted in the eukaryotic expression of core+1 protein in Huh7 cells and provided an appropriate tool for future studies on the potential interference of core+1 with intracellular signaling pathways and cellular protein interaction.

Although measles disease has controlled measles disease worldwide, there are some reports of the viral infection in vaccinated populations. Therefore, accurate and timely diagnosis is essential for the virus. Most of new molecular methods for virus detection cannot quantify the virus load. The purpose of this study was to set up Real-time PCR method using measles virus F gene. Firstly, two primers and a probe from the F gene cloned into plasmid pET-22b (+)(Novagen) were designed. A ten fold serial dilution of standard plasmid containing the gene was prepared and a standard curve was plotted. The project was carried out on the 7500 Real-Time PCR detection System (Applied Biosystems) with Taq man kit (Applied Biosystems). At least 30 viral particles per reaction could be determined and the dilutions between 10-4 to 10-9 equivalent 3×101 to 3×106 copies of the standard curve) were linear at best. Real-time PCR results showed that the technique can be used as highly specific and rapid method to detect and to determine the number of measles viral particles from the samples containing the virus.

Cryptococcus neoformans is opportunistic capsulated yeast that infects immunocompromised individuals. Infection occurs due to inhalation of the agent and common infection form is meningitis. Several methods are common to detect this life threthening agent but none of them have well accepted in sensitivity, specificity and rapidness. In this research the effort has been on installation and optimizing the LAMP assay in order to detect the agent itself and its distribution importance of Cryptococcus neoformans in serum specimens of AIDS patients. Atotal number of 34 serum specimens were collected from 34 AIDS patients. After applying sensitivity and specificity tests for the installed technique, optimization of the assay was performed on the specimens. The specimens got inactivated and after steps of DNA extraction, 6 designated primers for the target gene (ura5) were applyed for detection of Cryptococcus neoformans by using the LAMP method. In this study 3 specimens of the total number of 34 AIDS patients were reported positive by using LAMP technique. This it reveals the importance of Cryptococcal infection in AIDS patients.Conclution: Loop-mediated isothermal amplification is an applicable, sensitive, quick and lucrative method for detection of Cryptococcus neoformans. Diagnostic laboratory can use this method without requiring any special instruments.

Listeria bacteria are widespread and commonly found in soil, sewage, dust and water. Listeria monocytogenes and the other Listeria species have been isolated from a variety of raw and processed foods as well. The objective of this study was to determine the prevalence of Listeria spp. in retail stores located retail raw milk, traditional cheese and ice-cream in Shahrekord and Shiraz. A total of 178 samples of raw cow milk (n=45), raw goat milk (n=32), traditional cheese (n=41) and traditional ice-cream (n=60) collected randomly. All the samples were evaluated for the presence of Listeria spp. by using standard cultural methods, then confirmed with Polymerase Chain Reaction (PCR). Based on conventional bacteriologic tests, 24 of 178 samples (13.5%) were positive for Listeria spp. The prevalence of Listeria in raw cow milk, raw goat milk, traditional cheese and traditional ice-cream were 11.1%, 3.1%, 24.4% and 13.3%, respectively. The most species isolated was L. innocua (62.5%) and the others were L. monocytogenes (37.5%). Our results indicate the potential risk of infection with Listeria among people who consume raw and unpasteurized dairy products. Further intensive studies is suggested to evaluate of the prevalence of Listeria spp. in the other food products especially ready to eat foods.

Chlamydophila psittaci is a lethal intracellular bacterial species that causes endemic avian chlamydiosis, epizootic outbreaks in mammals, and respiratory psittacosis in humans. Chlamydia psittaci is a gram negative bacterium that can be transmitted from pet birds to humans. It is known that pigeons, like many other bird species, can harbor Chlamydia psittaci. The study aimed to determine the molecular frequency of Chlamydia psittaci in feces of pigeons in Chaharmahal Va Bakhtiari province using PCR technique. 300 feces samples of pigeons were collected from Chaharmahal Va Bakhtiari province's townships. Genomic DNA was extracted directly from specimens. PCR was performed using specific primers for investigation of ompA gene of Chlamydia psittaci. The analyses demonstrated a high frequency of Chlamydia psittaci (14.33%) among the tested samples. The highest and lowest frequencies of the bacterial infection were observed in Kiar and Lordegan cities with 16.66 and 8%, respectively. The results of the present study indicated that Chlamydia psittaci infections are highly prevalent amongst pigeons of Chaharmahal Va Bakhtiari province. According to these findings, examination of pigeons and wild birds for control and prevention of the distribution of this pathogen it seems to be necessary in order to control the infectious agent and then, to prevent the economic losses and health hazards.

Diarrhea in newborn calves caused by enterotoxigenic Escherichia coli K99 is one of the important problems is the animal industrial husbandry. In this study we try to find the frequency of E. coli k99 in diarrheal newborn calf, and also determine antibiotic susceptibility of isolates.

312 rectal swabs of diarrheal calves were obtained in various parts of Fars province. The samples were investigated using biochemical test. All E. coli isolates tested for antibiotic susceptibility using disc diffusion method.

of 312 samples, 298 E. coli isolates were obtained, from which the slide agglutination method 13 isolates were identified Escherichia coli k99. These isolates were susceptible to enrofloxacin, nalidixic acid and flomocuen antibiotics. Also, there is some wide range of antibiotics resistance to in Escherichia coli k99.

The prevalence of Escherichia coli k99 with diarrhea in calves was 4 / 3% in Fars province which compared to other reports ranking in the lower range.

Canola disease is one of the most important challenges in canola production. In different countries, various fungal agents, such as Rhizoctonia solani Pytium sp., Phytophthora sp. and Fusarium sp. have been introduced as root rot and damping off agents of canola. The aim of this study was to identify the fungal agents involving in canola foot and root rot and, to determine the most frequent fungal agent of the infection in Marvdasht region (Fars province). In this study, root and foot rotted plants were collected and their fungal agents were isolated. Sampling was performed in various fields in Marvdasht. Isolated fungi were cultured on PDA and purified on 2% water agar. Identification of fungal specious was carried out by using standard keys. All purified isolates were tested for pathogenicity. In this study, five pathogenic fungi belonging to four genera, Fusarium Rhizoctonia Phoma and Pythium were isolated from diseased samples. Among all isolates, F. solani (25%) and Phoma betae (2.5%) had the highest and lowest frequency, respectively. R. solani was very aggressive, whereas P. aphanidermatum and F. solani had produced very mild disease symptom. Our findings showed that canola root rot caused by R. solani is wide dispersed in Marvdasht region in early growth stages; so we strongly recommend that R. solani anastomosis group be identified in the next study. Moreover, usage of anti-microbial resistance and tolerance variety might be investigated. This is the first report of R. solani existence on canola root in Fars province.

Rhizobacteria are the most important microorganisms in the soil that have major role in fixing nitrogen than other soil microorganisms. The aim of this study is using native rhizobium of Fars province calcareous soil and determination the role of urea fertilizer in giving high yield and efficiency of alfalfa. First, native rhizobium is separated from alfalfa root nodules using YAM environment containing Congo red, and then it is identified with staining, and biochemical tests. This research is performed as a randomized plan and under different nutritional conditions, including fertilizer without urea, 100mg/kg urea fertilizer, 200mg/kg urea fertilizer, 1% animal manure, 3% animal manure and also under different microbial inoculations, including without bacteria, native rhizobium and standard rhizobial strain in six repeats in open air. The best growth is observed in the pots treated with local rhizobium. In comparing to urea fertilizer, the pots fertilized with 3% animal manure treatments showed a better growth. The most height plants were seen in the pots fertilized with local rhizobium treatments. The best treatment to give high yield was achieved with local rhizobial strain and 3% animal manure. The most active root nodules in nitrogen fixation were seen in the plants treated with local rhizobium. By comparing the native and standard rhizobial strains and according to calcareous soil of Fars province and hot and dry weather of this region, it is suggested that fertilization with local rhizobial strain and animal manure can result in high yields and nitrogen fixation in the root nodules of the plant.