سهیلا مرادی بید هندی

Leptospirosis is a common zoonotic infectious disease, that cause by pathogenic Leptospires. FlaB2 is a periplasmic flagellum protein that is expressed during infection in the host. This protein is conserved among all pathogenic Leptospiral serovars. The aims of this study was molecular evalution of the gene encoding FlaB2 protein by PCR method in pathogenic Leptospira serovars in Iran. In this study, 20 pathogenic Leptospira serovars and two nonpathogenic Leptospira serovars were used. All 22 Leptospira serovars were cultured into the selective medium EMJH and genomic DNA was purified using standard phenol-chloroform method. Specific primers were designed for amplification of flaB2 gene and its sensitivity and specificity were evaluated by PCR. The results of PCR product of flaB2 gene was 1050 bp and it was observed that all 20 pathogenic leptospira serovars contained the flaB2 gene. This gene was not amplified from two non-pathogenic L. biflexa. The results of this study showed that the gene flaB2 gene was present only in pathogenic serovars. Therefore, molecular characterization of flaB2 gene is important for detection of pathogenic serovars from nonpathogenic Leptospira serovars

 Leptospirosis is one of the most common zoonotic diseases worldwide, occurring mostly in tropical, subtropical, temperate, and humid regions with heavy rainfall. It is important to diagnose this condition correctly and promptly. Loa22 is an outer membrane protein exposed to the surface in some Leptospira serovars. The purpose of this study was to determine the presence of the gene encoding Loa22 protein in Leptospira interrogans serovars.

 The present study was conducted on 23 pathogenic leptospira serovars and two non-pathogenic leptospira serovars. These serovars were prepared from the Reference Laboratory for Leptospira, Department of Microbiology, Razi Vaccine, and Serum Research Institute, Karaj, Iran. After genomic DNA extraction using the standard phenol-chloroform method, loa22 gene was amplified by specific primers.

PCR was performed on the loa22 gene by producing a 671-bp fragment. The results showed that the loa22 gene was present in all 23 pathogenic leptospira serovars but not in the non-pathogenic L. biflexa. The specificity of tested primers was confirmed as well.

 The loa22 gene is a specific gene for pathogenic leptospira serovars that is not found in saprophytic serovars, so it is suggested that this gene be used to detect leptospira pathogenic serovars.

 Salmonellosis is an important infectious zoonotic disease that makes it even more significant to identify and control the causative strains. Molecular methods, especially polymerase chain reaction (PCR), for virulence genes can help to quickly and accurately identify Salmonella strains. Accordingly, the purpose of this study was molecular identification based on sivH, hilA and sefA genes and serotyping of Salmonella strains isolated from livestock in Alborz province, Iran.

 The present study was conducted on 30 Salmonella strains isolated from livestock in Alborz province. Salmonella strains were isolated using morphological identification and differential and selective culture media. DNA was then extracted by boiling, and PCR was performed to detect the virulence genes of hilA, sivH, and sefA. The sensitivity and specificity of the primers used were determined using PCR.

The PCR findings showed that 27 (90%) isolates had the hilA gene, 10 (33.3%) isolates had the sefA gene, and 24 (80%) isolates had the sivH gene. Moreover, the highest frequency among serotypes was related to Salmonella typhimurium (10%). The sensitivity of ST11-ST15, hilA, sefA, and sivH primers were estimated at 0.0001, 1, 0.1, and 0.001 ng/mol, respectively. The specificity of primers for Salmonella strains was also confirmed.

 Identifying livestock with salmonellosis and isolating pathogenic strains from other livestock are of the most important methods capable of reducing the prevalence of foodborne infection in consumers. This can be achieved by the PCR technique for virulence genes, especially hilA, which is more prevalent among Salmonella strains.

Different species of Salmonella are important pathogenic bacteria in animals and humans that often infect poultry flocks. Most Salmonella infections in humans are caused by eating foods contaminated with this bacterium. The aim of this study was to determine the Salmonella infection status of several poultry farms in Khuzestan province and to determine the serotypes and antimicrobial resistance pattern of isolated Salmonella. In the year 1392 total of 1829 samples including stool swabs, litter samples, feathers and eggshells were collected from a broiler mother farm, two laying mother hen farms and an incubator and cultured in enriching and selective media and detected using biochemical tests according to standard methods. In this study, 10 Salmonella isolates were isolated including serotypes enteritidis (3 cases), corvallis (3 cases), typhimurium (3 cases) and ruzizi (1 case) using Salmonella serum type tests. After performing antibiogram test with standard disk diffusion method, it was determined that 100% of the isolates were resistant to Colistin, Cefalexin and Cefazolin. Isolates were highly resistant to Ceftazidime, Nalidixic acid, Ampicillin, Ciprofloxacin, Nitrofurantoin, Amoxicillin, Neomycin, Streptomycin, Kanamycin, Ceftizoxime, Gentamicin and Tetracycline. All Salmonella isolates in this study were sensitive to Amikacin, Enrofloxacin, Ceftriaxone, Imipenem, Cefixime, Cefotaxime, Ofloxacin, Cefepime, Piperacillin, Florfenicol, Furazolidone, Chloramphenicol and Trimethoprim- Sulfamethoxazole.

The expression of most genes involved in pathogenesis in Pseudomonas aeruginosa is controlled and regulated by a gene system called quorum sensing (QS). The purpose of this study was to investigate the quorum quenching of garlic extract in preventing the expression of genes involved in QS system of P. aeruginosa.

In the present cross-sectional study, 12 P. aeruginosa strains were collected from burn wounds, cultured on special media and then confirmed by differential tests. The PCR test was performed to detect the lasI and lasR genes. The expression level of lasI gene was assessed in the presence of garlic extract and tobramycin antibiotic by real-time PCR, and the active ingredient of garlic extract was analyzed by HPLC.

Examination of 12 strains cultured on special and differential media showed that all 12 strains were P. aeruginosa. The PCR results indicated that all strains had 100% lasI and lasR genes. The real-time PCR results also revealed that the garlic extract was able to reduce the expression level of lasI gene in P. aeruginosa. It was found that tobramycin has a greater ability to reduce the expression level of this gene compared with garlic extract.

This study showed that the garlic extract could reduce the expression of lasI gene in P. aeruginosa isolates, and that the active ingredients of this extract could be used to treat infections as an alternative to antibiotic therapy.

Due to their antibacterial effects, herbal remedies have an important role in the treatment of infections; particularly in infected wounds. Increased bacterial resistance to antibiotics combined with increased side effects related to their administration has led many researchers to investigate alternative herbal treatments in recent years. In line with this trend, the objective of this study was to assess the antimicrobial effects of Dr Kamkar’s cream on different microbial strains in vitro.

The susceptibility of isolates to the cream was evaluated using well diffusion, the Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) methods. 11 microorganisms were analyzed which included 5 strains routinely used to assess antibacterial effects and 6 clinical isolates were obtained from patients’ wounds. Based on CLSI (2018) standards, the Kirby& Bauer method was utilized to investigate the sensitivity of the strains to commonly used antibiotics.

The herbal cream was effective against 10 strains, while only 1 strain demonstrated resistance. Bacillus subtilis was the most susceptible bacteria to the cream with a zone of Inhibition of 31mm, followed by Bacillus anthracis (25mm), Staphylococcus aureus (22mm), Acinetobacter baumannii (22mm), Staphylococcus epidermidis (20mm), Bacillus cereus (16mm), Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus (14mm), Streptococcus pyogenes and Candida albicans (20mm) and resistant to Escherichia coli.
MIC findings indicated that the herbal cream had maximal effect against Bacillus cereus, and minimal efficiency against Streptococcus pyogenes and Candida albicans, while MBC results showed maximal effect against Bacillus cereus and minimal effect against Pseudomonas aeruginosa.
 Results of antibiograms revealed resistance to several antibiotics.

According to the results, Dr Kamkar’s herbal cream has suitable antimicrobial activity against certain pathogenic microorganisms and can be used alone or in combination with Antibiotics in the treatment of infected wounds.

Klebsiella pneumoniae is one of the most important causes of nosocomial infections, especially wound infection after surgery. One of the mechanisms of antibiotic resistance in K. pneumoniae strains, especially ciprofloxacin, is the presence of efflux pump.

The aim of this study was to evaluate the anti-efflux activity of Grammosciadium platycarpum Boiss. & Hausskn. extract on the expression of oqxA efflux pump in K. pneumoniae strains which were resistant to antibiotics.

In this experimental study, clinical specimens were collected from hospitals in Tehran and the K. pneumoniae strains were isolated. Subsequently, ciprofloxacin-resistant strains containing the oqxA efflux gene were detected using PCR. Finally, the gene expression of oqxA efflux pump in the strains treated with G. platycarpum extract was investigated using Real Time PCR.

In this study, 50 K. pneumoniae strains were isolated from clinical specimens and the results of antibiotic susceptibility showed that 70% (35 strains) of isolates were resistant to ciprofloxacin and oqxA gene was observed in 43% (15 strains) of ciprofloxacin resistant K. pneumoniae strains. Moreover, Real Time PCR results showed that the expression of oqxA gene in the strains which are treated with extract, down-regulated significantly.

The results of this study showed that the G. platycarpum extract can inhibits the expression of the oqxA efflux pump in K. pneumoniae strains, and with further studies, the G. platycarpum extract can be used as a candidate for the drug design.

Leptospirosis is one of the most important diseases Transferable from animal to human. LipL32 is one of the most important outer membrane proteins that exist only in pathogenic Leptospira spp., which is a suitable factor for the detection of pathogenic Leptospira. Therefore, the aim of this study is the molecular detection of pathogenic Leptospira based on lipL32 gene. Saprophytic and pathogenic Leptospira serovars were used in this study. The bacteria were inoculated into the selective culture medium and extraction of the genomic DNA was performed by standard Phenol-Chlorophorm method. The specific primers for proliferation of lipL32 gene were designed. In terms of annealing temperature was 61°C, suitable concentration 10 pmol, sensitivity was -2 11 pg/ μl and its characterization in PCR were investigated. According to PCR data, it was confirmed this gene was present only in pathogenic Leptospira serovars and 272 bp fragment indicating the replication of the lipL32 gene and was not observed in non-pathogenic species. Identify methods for rapid detection of pathogenic leptospires before entering the acute phase of the disease is very important. The results showed that PCR using lipL32 gene was high specificity and sensitivity. So for the detection of pathogenic Leptospires, molecular methods based on PCR using lipL32 gene in the shortest possible time offer accurate and reliable response is recommended.

Bacterial cellulose synthesized by some microorganisms, including Acetobacter xylinum, has been widely used in various industries due to its specific properties. The purpose of this study was to optimize the cultivation condition for the production of microbial cellulose in a new culture medium.   Materials & In this experimental study, new sources of carbon and nitrogen were added to the Hestrin-Schramm medium, containing A. xylinum, and incubated for 7 days under static conditions. Carbon sources included glucose, galactose, fructose, lactose, sucrose, maltose, ethanol, methanol, inositol, glycerol, xylose, and mannitol and nitrogen sources included ammonium sulfate, ammonium nitrate, sodium nitrate (1, 3, 6, 9  g/l HS medium), peptone and yeast extract (5, 10, 15, 20  g/l  HS medium). Sodium alginate and sodium acetate were used to investigate the viscosity effect and to adjust the medium pH. Scanning electron microscopy, X-ray diffraction, and FTIR spectroscopy technique were used in order to confirm the cellulose production. Sodium alginate and sodium acetate were used to investigate the viscosity effect and determine the pH adjustment. Scanning electron microscope, X-ray diffraction and FTIR spectroscopy technique were used in order to confirm the cellulose production.   Four carbon sources including glycerol (without a significant drop in pH), glucose, fructose, and inositol produced the highest amount of cellulose, respectively. Organic nitrogen sources, particularly peptone, had a great impact on cellulose production, unlike mineral nitrogen sources. The optimum amount of sodium alginate as the viscosity agent and sodium acetate as the buffer was 1.2 and 3 gram per liter of culture medium, respectively. X-ray diffraction showed the highest crystallinity index in medium containing glucose, fructose, inositol, and glycerol, respectively. The amount and intensity of infrared absorption in FTIR scanning of the products of culture media containing glucose and glycerol and comparing them with other similar cellulose graphs confirmed the cellulose production. Furthermore, Scanning Electron Microscopy studies clearly showed a nanofiber structure of microbial cellulose in media with better carbon sources.   According to our findings, glycerol and peptone have the most impact on microbial cellulose production. It was also indicated that addition of 2.1 g/ L sodium alginate to the culture medium as the viscosity agent along with pH control during the process by adding 3 g/ L sodium acetate can have a significant effect on cellulose production.

Salmonellosis is a common disease among human, animal and poultry which is raised as a food-borne illness. One of the most common serotypes of this bacterium is salmonella infantis. The aim of this study was to investigate the antimicrobial resistance of 70 salmonella infantis isolates from poultry between 1389-1390 in Arak. In order to confirm the genus of Salmonella, all isolates showed 1.5 Kb band on agarose gel. The results of antibiogram that was performed by the Kirby-Bauer disk diffusion method according to Clinical Laboratory Standards Institute (CLSI) showed that all isolates (100%) were resistant to nalidixic acid and nitrofurantoin. Among 70 isolates, 2 (2.9%) cases were resistant to 11 antibiotic followed by 10 (14.3%) to 10 antibiotic, 8 (11.4%) to 9 antibiotic, 6 (8.6%) to 8 antibiotic, 22(31.4%) to 7 antibiotic, 12(17.1%) to 6 antibiotic and 8(11.4%) to 5 antibiotic. Also twenty-nine antibiotic resistance patterns were found.

Etant donnée la circulation des souches de Bordetellapertussisau sein des populations avec une couverture vaccinale élevée, une bonne compréhension de la nature de cette bactérie provoquant la coqueluche est nécessaire. Une variété de techniques ont été mises en place afin de faciliter la caractérisation des souches de Bordetellapertussisau sein des différentes populations. Une analyse génotypique a été menée sur une collection de deux souches vaccinales utilisées entre les années 2000 et 2014dans la production de vaccins inactivés de la coqueluche à l’Institut de Recherche sur les Vaccins et les Sérums Razi (Karaj, Iran). Au total, 10 isolats cliniques et 2 souches de références (Tohama 1 et 18323) ont été analysés par électrophorèse en champ pulsé (ECP). Selon nos résultats, le profil génétique de la souche-mère et des semences actives ne montre aucun changement significatif dans la fréquence des types d’empreintes observée avec les souches vaccinales. Ceci reflète donc la bonne homogénéité des profils étudiés. De plus, un sérotypage a été effectué avec des antisérums monoclonaux des agglutinogènes 2 et 3. L’analyse des fimbriae a révélé que les 10 isolats cliniques exprimaient Fim3.

Excessive use of antibiotics and biocides has led to emergence of resistant Staphylococcal strains. The aim of this study was to examine resistance of Staphylococcal strains biofilm isolated to biocidal components.
100 clinical isolates of Staphylococcus aureus were collected from hospitalized patients with infectious skins from two Shohadaye Tajrish and Valiasr hospitals for one year in Tehran City. Isolated strains were identified by standard biochemical tests and confirmed using polymerase chain reaction. The pattern of resistance of strains to antibiotics and biocides such as Savlon, Decosept and Deconex (Dermocept) were determined by disc diffusion method and their minimal inhibition and cidal concentrations were estimated using microdilution. The biofilm of MDR strains were formed on polystyrene microplates.
Most of strains were resistant to penicillin, amoxicillin, ampicillin and methicillin and the most sensitivity was seen to clarythromicin. The phenotyping findings of biofilm formation show that 6%, 23.2% and 50.4% of isolates were able to biofilm formation as strong, intermediate and weak, repectively, and only 20.4% were unable to form biofilm. Biofilm had the lower and higher resistant to Savlon and Deconex, respectively.
The prevalence of hospital resistance strains with ability of biofilm formation can be serious danger for health of society in an extended spectrum of patients.

Salmonella infections are the second leading cause of zoonotic bacterial foodborne illness. Main source of infection in human is contaminated food products. The aim of this study was sub typing isolates of Salmonella entericaobtained during our previous study byPulsed Field Gel Electrophoresis (PFGE) technique. All 46 Salmonella isolates were serotyped and then subjected to PFGE. Total isolates were analyzed by means of the molecular technique XbaI PFGE. In this study, PFGE and serotyping were used to subtype 46 Salmonella isolates belonging to 27different serovars and derived from human and different food origins. Among these isolates, S. Typhimurium was found to be the most predominant serovar. 40 PFGE patterns out of 46 isolates were obtained. The Discrimination Index obtained by serotyping (DI = 0.93) was lower than PFGE (DI = 0.99). Subtyping of Salmonella enterica is very important and shows that animal origin can be one of a reservoir that potentially could be transferred to human through the food chain. In addition, results of this study also revealed that this procedure is a golden standard for genotyping of such salmonellaserotypes.

Salmonella enterica is recognized as one of the major food-borne pathogens with more than 2,500 serotypes worldwide. The present study addresses antimicrobial resistance of Salmonella enterica serovar Enteritidis isolates in Iran. A collection of 151 Salmonella spp. isolates collected from poultry were serotyped to identify Salmonella Enteritidis. Sixty-one Salmonella Enteritidis were subsequently tested against 30 antimicrobials. A high frequency of antimicrobial resistance was observed against nitrofurantoin (n=55, 90.2%) followed by nalidixic acid (n=41, 67.2%), and cephalexin (n=23, 37.7%). Multi drug resistance were observed in 35 (57.4%) out of 61 isolates. Twenty-six antimicrobial resistancepatterns were observed among the 61 Salmonella Enteritidis. All isolates were susceptible to ofloxacin, imipenem, enrofloxacin, chloramphenicol, gentamicin, and 3rd and 4th generation cephalosporins. In conclusion, our results revealed that implementing new policies toward overuse of antimicrobial drugs in Iranian poultry industry are of great importance.

Leptospirosis is a zoonotic disease with global distribution that caused by pathogenic spirochetes of the genus Leptospira. Accurate diagnosis for differentiation of leptospirosis from other pyrogenic infections prevailing in the same locality and is imperative for proper treatment. Therefore a molecular diagnostic test with high specificity and sensitivity such as PCR is essential. Gene encoding of outer membrane proteins of Leptospira are potential candidates that may be useful as diagnostic and analysis of the disease. In this study, lipL21 gene was used for detection and differentiation of pathogenic from saprophytic leptospiral serovars in PCR assays. The leptospiral lipL21 gene expressed only in pathogenic Leptospira spp. The bacteria were inoculated into the EMJH (with 5% rabbit serum) and extraction of the genomic DNA was done by standard Phenol-Chlorophorm method. The specific primers for proliferation of lipL21 gene were designed. The lipl21 gene was observed in pathogenic leptospira and was not in saprophytic leptospires. The specificity and sensitivity of PCR was evaluated. PCR assay with high specificity and sensitivity may prove to be a rapid method for diagnosing acute leptospirosis and designed a positive control to optimize this diagnostic test. The results showed that molecular detection of pathogenic leptospiras based on lipL21 gene can be used for laboratory diagnosis of leptospirosis.

Leptospirosis is a zoonotic disease in humans and animals, caused by the bacterium Leptospira interrogans. Gene expressing LipL21 is one of the genes identified in the bacterium, existing only in the pathogenic strains. The aim of this study was to cloning and analyzing the sequence of the gene encoding surface lipoprotein, LipL21, in five vaccinal leptospira serovars in Iran. Pathogenic Leptospira interrogans serovars were cultured in EMJH medium with 10% rabbit serum. After genomic DNA extraction, PCR with specific primers was employed and the resulting product inserted in a vector then transferred into E. Coli DH5&alpha. The recombinant plasmids were finally sent for sequencing. The analysis of gene lipL21 in domestic vaccinal serovars and comparison of them with other serovars in the GenBank database revealed that three vaccinal serovars serjo hardjo, canicola and pomona had 100% similarity with each other and grippotyphosa serovar had the highest difference with the vaccinal serovars. In general, the results showed that this gene is a highly conserved gene in the domestic vaccinal serovars and serovars in the GenBank database with more than 95.7 percent similarity. These results showed that the gene, lipL21, is highly conserved in the vaccinal serovars (similarities > 96.4 %). Therefore, the gene encoding surface protein LipL21 can serve as a useful serologic test with high specificity and sensitivity for diagnosis of leptospirosis in clinical samples and in future as an effective subunit vaccine candidate to be used.

Salmonella infection is a major public health problem in the world and in most parts of the world, particularly in developing countries are of utmost importance. Poultry, human, animal and food play an important role in transmission, economy and public health aspects. Yet studies show that Salmonella enteritidis and Salmonella typhimurium is the predominant serotypes. Due to limitations in isolation in laboratory, using molecular techniques as a rapid, inexpensive, specific and sensitive diagnostic must be used. The source of the infection could be important in the control strategies. Spread of resistance genes via plasmid among different serotypes of Salmonella is of great importance. Identification and prevention of drug resistance genes among serotypes of Salmonella could be one of the most important issues in the study of its ressistance. To determine the susceptibility patterns of isolates, the protocols of treatment can be modified and replaced with effective antibiotics.

Salmonellosis is a very important disease of avian species because of its huge economic impact، worldwide distribution and difficulty posed in its control. Fowl typhoid and pullorum disease، is caused by Salmonella enterica subsp enterica serovar Gallinarum biovar Gallinarum and Pullorum. The purpose of this study was to investigate the biochemical characteristics and antimicrobial susceptibility of Salmonella gallinarum and Salmonella pullorum. A total of 13 Salmonella isolates، identified by biochemical tests and specific antisera including Salmonella gallinarum (n=10) and Salmonella pullorum (n=3). All were found to be susceptible to gentamicin. Also 7 (53. 8 %)، 6 (46. 1%) and 5 (38. 4%) isolates were resistant to streptomycin، cephalexin and nalidixic acid respectively. Multidrug resistance to three or more antibiotics was observed in 6 (46. 1%) isolates and overall 9 antibiotic resistance patterns were recorded. The results showed that poultries as a source of antimicrobial resistance could pose a serious risk to public health via food chain transfer. Hence more epidemiological surveillance programs and antibiotic susceptibility investigations are advised.

Leptospirosis caused by infection with pathogenic leptospires، which is the most prevalent zoonotic disease in the world. The outer membrane proteins (OMPs) of pathogenic leptospires such as LipL41 play a crucial role in pathogenesis of this disease. Therefore a major challenge to develop an effective vaccine against leptospirosis is application of basic research on the OMPs of leptospires to improve vaccine development. The aim of this study was cloning and analyzing of the lipL41 gene from vaccinal serovars of leptospires in Iran، in order to identify genetic conservation of this gene. Three vaccinal serovars of Leptospira were used in this study. The lipL41 gene of these serovars were amplified and cloned in the pTZ57R/T vector. The recombinant clones were confirmed by colony-PCR and sequencing. The sequenced genes were analyzed for their homology between them and other submitted sequences in Genbank database using the BLAST and MegAlign program. PCR amplification of the lipL41 gene resulted in the 1065 bp gene product in vaccinal serovars tested. In our study، nucleotide sequencing results showed high similarity (>94%) within the leptospiral vaccinal serovars. The genetic conservation of the lipL41gene among different serovars of Leptospira indicated the capacity of utilization of this gene for development of recombinant vaccine against leptospirosis.

The genus of Salmonella is very polymorphic and comprised of a number of genetically closely related serotypes. It is one of the emerging pathogen in food-borne disease which is often found in contaminated chicken eggs. Salmonella enterica is considered one of the major pathogens in public health worldwide. A total of 31 Salmonella isolates identified by specific antisera، which included Salmonella enteritidis (51. 6%)، Salmonella typhimurium (25. 8%)، Salmonella infantis (19. 4%) and Salmonella colindale (3. 2%). DNA was extracted using phenol- chloroform- isoamylalchol method. All the isolates showed fliC gene (1500bp) by using specific primers. PCR products were subjected to digestion using HhaI restriction endonuclease. PCR- RFLP results showed 3 patterns between all isolates. Our research gained in this study demonstrated that using HhaI restriction endonuclease could differentiate Salmonella enteritidis and Salmonella colindale but there is similarity between pattern of Salmonella typhimurium and Salmonella infantis.

Leptospirosis is a zoonosis infectious disease that is prevalent in tropical and subtropical regions and is caused by the pathogenic serovars of leptospires. Hence, we aimed at investigating the prevalence of antibodies against these bacteria in the blood samples of suspected leptospirosis. the human serum samples (N = 130) were obtained from patients clinically suspected leptospirosis. The Serum level of IgM antibodies were studied by ELISA kit (PrioCHECK) in Razi Vaccine and Serum Research Institute (Karaj), 2011-2012. Anti-leptospira IgM class was observed in 21(16%) samples. The relative distribution of the disease was reported in men (80.95%), women (19.04%), and farmers (30.95%) and in 20-40-year group (57.14%). Contact with contaminated water was the most common cause of infection (52.38%) and fever was the most common sign of Leptospirosis (72.2%). Due to the occurrence of anti-leptospira antibodies in 16% of suspected cases, it is recommended that routine ELISA be done at least in major diagnostic centers.

Leptospirosis is one of the most important zoonosis with worldwide distribution. lipL41 is an immunogenic outer membrane protein found in pathogenic Leptospira species and may be used in diagnostic method and also can be a good candidate for recombinant vaccine against leptospirosis. The aim of this study was designing a positive control for molecular diagnosis of pathogenic Leptospira spp. by PCR based on lipL41 gene. Five pathogenic serovars and one saprophytic species were used in this study. The serovars were subcultured into the selective culture medium (EMJH) and then the genomic DNA extracted. The lipL41 gene amplified using the specific primers. The PCR product were ligated in pTZ57R/T vector and transformed in competent E. coli Top10 cells. The confirmation of the recombinants was made by picking the white colonies and carrying out colony PCR amplification of the gene. The recombinant plasmids were extracted using a commercial extraction kit. PCR amplification of the lipL41 gene using the specific primers resulted in a 1065 bp in all five pathogenic serovars tested. No PCR products were amplified from the non-pathogenic L. biflexa. Positive colonies plasmid vector was isolated from cells by kit. PCR test was carried out with positive control and PCR products were observed on gel electrophoresis. Due to slow growth of Leptospira، and because of limited diagnostic capacity using a rapid and accurate molecular tests like PCR with a positive control to confirm its accuracy is essential. Therefore، the cloned lipL41 gene in this study that has been prepared in Leptospira reference laboratory can be used as a positive control in all laboratories using the PCR test.

Leptospirosis is an infectious bacterial disease caused by pathogenic serovars of Leptospira. Development of reliable and applicable diagnostic test and also recombinant vaccine for this disease require specific antigens that are highly conserved among diverse pathogenic leptospiral serovars. Outer membrane proteins(OMPs) of leptospira are effective antigens which can stimulate remarkable immune responses during infection, among them LipL41 is an immunogenic lipoprotein which is present only in pathogenic serovars so it could be regarded as a good candidate for vaccine development and diagnostic method. In order to identify genetic conservation of the lipL41 gene, we cloned and sequenced this gen from Leptospira interrogans serovar vaccinal and field of Grippotyphosa. Leptospira interrogans serovar vaccinal Grippotyphosa (RTCC2808) and serovar field Grippotyphosa (RTCC2825)were used to inoculate into the selective culture medium(EMJH). The genomic DNA was extracted by standard phenol-chloroform method. The lipL41 gene were amplified by specific primers and cloned into pTZ57R/T vector and transformed into the competent E. coli (Top10) cells. the extracted recombinant plasmid were sequenced. And the related sequences were subjected to homology analysis by comparing them to sequences in the Genbank database. PCR amplification of the lipL41 gene resulted in the 1065 bp PCR product. DNA sequence analysis revealed that lipL41 gene between serovar vaccinal Grippotyphosa (RTCC2808)and serovar field Grippotyphosa (RTCC2825) in Iran was 100%. It was also showed that the lipL41 gene had high identity (96%-100%) with other pathogenic serovars submitted in Genbank database. The results of this study showed that the lipL41 gene was highly conserved among various pathogenic Leptospira serovars(>95.9 % identity). Hence the cloned gene could be further used for expression of recombinant protein for serodiagnosis and also can be a good candidate for vaccine against leptospirosis.

Leptospirosis is an emerging infectious disease and is considered to be the most widespread zoonotic disease in the world. LigB is an immunogenic outer membrane protein. The leptospiral ligB gene expressed only in pathogenic Leptospira spp. The aim of this study was molecular diagnosis of pathogen Leptospires by PCR based on ligB gene. Five pathogenic Leptospires: L. canicola, L. grippotyphosa, L. pomona, L. icterohaemorrhagiae, L. serjoe hardjo and saprophytic L. biflexa were used in this study. The bacteria were inoculated into the selective culture medium and extraction of the genomic DNA was performed by standard Phenol-Chlorophorm method. The specific primers for proliferation of ligB gene were designed. The specificity and sensitivity of PCR method was evaluated. PCR product was 1041bp which indicated proliferation of ligB gene which was supported using electrophoresis. The PCR based on ligB gene detected all pathogenic reference serovars of Leptospira spp. tested. No PCR products were amplified from the non-pathogenic L. biflexa. Considering the spread of Leptosperosis in moderate and hot areas which have high rate of fall, a proper molecular diagnostic test with high specificity and sensitivity such as PCR is essential. PCR assay with high specificity and sensitivity may prove to be a rapid method for diagnosing acute leptospirosis. The results suggested that the PCR based on ligB gene can be used for detection of pathogenic leptospires.

RFLP-IS6110 standard technique to genotyping M. tuberculosis.The aims of this study were to identify the genetic diversity of M. tuberculosis population in Markazi province and to recognize the mode of disease transmission in this region. The RFLP results of 42 isolates of M. tuberculosis deposited in the Mycobacterial Centre from Markazi province were analyzed. DNAs isolated from these isolates were enzyme digested with Pvu II, and hybridized with a PCR amplified DIG-labeled IS6110 probe. The isolates were classified into four groups, based on the copy numbers, as follows: (1) lacking IS6110 element; (2) low copy numbers (1-2); (3) intermediate copy numbers (3-5); and (4) high copy number (6-17). Copy numbers higher than 17 however were not observed in any of the isolates studied, 72 percent of the isolates showed high copy numbers of IS6110, 13 percent intermediate copy numbers, 10 percent low copy numbers, whereas 5 percent isolates lacked IS6110 element. IS6110 DNA fingerprinting assisted us to find epidemiological links between some TB cases, and this technique estimates from reactivation of latent infection transmission of the disease in Markazi province. The low rate of clustering indicates that tuberculosis among studied population is resulted mainly from reactivation of latent infection in this region.