[Construction of mutant WbkA gene in Brucella abortus S19 by overlap extension PCR]

Background: Causing site direct mutation can be one of the efficient methods to evaluate the characteristics and properties of various genes. Brucellosis is the most common zoonotic infectious disease that would cause great economic losses. Thus, recognition of pathogenic and immunogenic factors in the genus Brucella can lead to control this health problem

Objectives: Considering the importance of site direct mutation in identification of genome structure and numerous ways to achieve this goal, Overlap Extension PCR is introduced as an improved technique for the removal and replacement of the gene target

Methods: For this study, with two-step PCR using specific primers, upstream and downstream fragments from target gene and antibiotic resistance cassette from plasmid pET28a [+], were reproduced and were connected to each other. The resulting fragment was cloned in specific position of pBluescriptIISK[-] plasmid by the restriction enzymes. Then, the construction was transferred into the genome of Brucella abortus by electroporation method

Results: Fusion PCR product was obtained without any change in the nucleotide sequence and then it was cloned into pBluescriptIISK [-] plasmid, finally the construction was replaced and the target gene was deleted

Conclusions: The results of this study show that the Overlap Extension PCR is an optimized and modified technique to create mutations in the bacterial genome structure and can easily be used in the family Brucella

[Molecular identification of Salmonella Infantis isolated from backyard chickens and detection of their resistance genes by PCR]

This study aims at molecular identification of Salmonella Infantis isolated from backyard chickens and the detection of their antibiotic resistance genes. A total of 46 Salmonella-suspected samples isolated from backyard chickens of northern Iran were collected. Serotyping was done by the traditional method and then confirmed by PCR. Antimicrobial susceptibility of the isolates against 13 antimicrobial agents was determined by the standard disk diffusion method. There were 44 samples identified as Salmonella. Serotyping results showed that all 44 isolates belonged to serogroup C1 and serovar Infantis. The most resistance observed was to tetracycline and doxycycline [100%], chloramphenicol [79%] and florfenicol [72%]. The floR, catI, tetA and tetG genes were used for the detection of florfenicol chloramphenicol and tetracycline resistance. In order to identify the phenotypic resistance in strains which showed resistance genes by PCR, colony PCR and culture on plates each containing antibiotic was performed simultaneously. All the Salmonella Infantis resistant to florfenicol and chloramphenicol harbored floR and catI. None of the Salmonella resistant to tetracycline carried tetA or tetG. The result of colony PCR and culture in antibiotic medium confirmed the results of PCR and indicated phenotypic resistance in these samples.

[Gene disruption in Salmonella typhimurim by modified Lamda Red disruption system]

There are many techniques to knock out directed genes in bacteria, some of which have been described in Salmonella species. In this study, a combination of SOEing PCR method and the lamda Red disruption system were used to disrupt phoP gene in wild type and standard strains of Salmonella typhimurium. Three standards PCR and one fusion PCR reactions were performed to construct a linear DNA including upstream and downstream of phoP gene and Kanamycin cassette. As a template plasmid, we used pKD4 which carries kanamycin gene flanked by FRT [FLP recognition target] sites. The resulting construct was electroporated into prepared competent cells of S. typhimurium. The transformants colonies related to the standard strain appeared on the LB-Km-agar plates after incubation, but there was no colony on LB-Km-agar plates corresponding to the wild type strain. The failure in transformation of the wild type strain may be because of inflexibility of the lamda Red disruption system in this strain or its unique restriction-modification system. However, by this construct we are able to generate phoP mutant in many of the Salmonella species due to high homology of the phoP gene which exists in different species

Construction of an iss deleted mutant strain from a native avian pathogenic Escherichia coli O78: K80 and in vitro serum resistance evaluation of mutant

Colibacillosis, caused by different serotypes of avian pathogenic Escherichia coli [APEC], is one of the important diseases in poultry industry. The isolate O78 is the most prevalent serotype of APEC in Iran. One of the APEC virulence factors, increased serum survival [iss] gene, is related to serum resistance. The usual form of colibacillosis in avian is extraintestinal, and serum resistance is applied one way by APEC to reach internal organs; hence, it appears that the control of colibacillosis in poultry regarding the deletion of iss and the construction of a serum sensitive APEC strain is beneficial. Additionally, the knowledge about APEC serum resistance could be extended using mutant strains. The present study was an attempt to generate an iss mutant strain from native APEC-O78 strain |c|1378 and to study the level of serum resistance of native APEC-O78 strain c1378 in comparison with its mutant [APEC-O78 strain c1378|D|iss]. The lambda red recombinase system was utilized to delete iss gene in native APEC-O78 strain c1378. This strain was first transformed with the plasmid pkD46 to introduce the lambda red recombinase system and then the PCR product with sequence homology to the iss gene and a kanamycin resistance marker was transformed into the APEC-O78 strain c1378. Serum sensitivity of mutant and wild type strain was investigated by microtiter test. The generation of mutant was successful and the iss was replaced with kanamycin resistance cassette. Also, it was observed that the mutant was sensitive to serum. However, serum sensitivity of iss deleted mutant was not statistically different from its parents. Application of lambda red recombination could be a simple and useful technique for production of a precisely defined gene deletion. Also, there may be some genes that compensate the activity of iss gene

[Molecular characterization and phylogenetic analysis of feline hemotropic mycoplasmas]

Feline hemotropic mycoplasmas are parasites of erythrocytes and include three species, Mycoplasma haemofelis, Candidatus mycoplasma haemominutum and Candidatus mycoplasma turicensis. Diagnosis of the infection with these microorganisms can be carried out using conventional assays such as blood cytology. However, these assays have a low accuracy and a high rate of false-positive results due to the poor techniques and procedures and high occurrence of artifacts. Therefore, molecular techniques such as polymerase chain reaction [PCR] are better methods for the diagnosis of infections by these bacteria. The purpose of the present study was to evaluate Feline hemotropic mycoplasma prevalence and phylogenetic analysis in Tehran. Sixty cat blood samples were collected from veterinary clinics in Tehran from 2011 to 2012. Giemsa stained blood smears have been examined by the light microscopes and the positive samples were used for DNA extraction and PCR. Positive PCR samples were sequenced for the differentiation of bacterial species and phylogenetic analysis. Thirty-two samples were positive in direct examination from which two samples were identified as M. haemofelis by the PCR. No positive samples of C. M. haemominutum or C. M. turicensis were found in PCR. Phylogenetic analysis of the isolates showed that these isolates were more similar to the isolates from China and Thailand compared to those from other countries. This study is the first report of phylogenetic analysis of hemotropic mycoplasmas in Iran. Based on the high sequence similarity between Iran, China and Thailand isolates, it can be concluded that these bacteria possibly had the same origin

Construction of a recombinant vector for site-directed mutagenesis in Salmonella typhimurium

Among all common techniques in site directed mutagenesis, Lambda Red recombinase system has been widely used to knock out chromosomal genes in bacteria. In this method, there is always the risk of DNA Linear digestion by host's restriction enzymes that leads to the low frequency of recombination. To overcome this, we constructed a recombinant vector to disrupt phoP gene in Salmonella typhimurium. The SOEing PCR method and restriction enzymes were used to construct the vector. The resulting plasmid, pTAAZ92, contains a Kanamycin cassette with two long homologous arms flanking of the phoP gene. After electrotransformation of the pTAAZ92 into the Salmonella typhimurium, the phoP gene is replaced by the Kanamycin cassette through homologous recombination. According to the high homology of the phoP gene in many of Salmonella species the pTAAZ92 can be used to disrupt the phoP gene in most of these species

Molecular detection of Leptospira spp. in the urine of cattle in northern Iran

Leptospirosis is a zoonosis of worldwide distribution, caused by Leptospira interrogans and is considered as an emerging global public health problem. Transmission usually results from direct or indirect exposure to the urine or other body fluids of leptospiruric animals which may become a source of infection for human or other animals. Having a humid climate with plenty of annual rainfall, Guilan province is a suitable environment for maintaining Leptospira spp. Hence, early detection of Leptospira spp. in the host prompts control and protection, and the polymerase chain reaction [PCR] is a suitable method. The present report aimed to demonstrate the PCR analysis of bovine urine for detection of leptospiral DNA. A total of 98 urine samples were randomly collected from cattle bladder in Rasht abattoir of Iran and the presence of leptospiral DNA was assayed by PCR amplification of rrs [16S rRNA] gene and the results confirmed by nested PCR. Out of 98 urine samples in 42 samples leptospires DNA was identified with the frequency of 43%. The high presence of the organism in the urine of carriers is a serious threat to the dairy farms and to the public health which requires an effective control measure in the north provinces of Iran

[Chemical composition and anti-candida activity of Trachyspermum ammi essential oil on azoles resistant Candida albicans isolates from oral cavity of HIV+ patients]

Oral candidiasis, caused by Candida albicans, is one of the most common infections in immunocompromised patients, especially in HIV+ individuals. The aims of this study were to evaluate the susceptibility of C. albicans isolates to azole drugs and Trachyspermum ammi essential oil. Oral swabs were cultured from 70 HIV+ patients and In order to identify and confirm of C. albicans isolates, Chrom agar, Corn meal agar, germ tube production, carbohydrate assimilation, growth at 45[degree sign]C and PCR were performed. Sensivity to fluconazol, ketoconazole and clotrimazol were assessed by disc diffusion and also the effect of T. ammi essential oil was determined by disc diffusion and microdilution broth methods. The causative agent, in 50 patients with oral candidiasis, was C. albicans [71.4%]. In sensivity determination survey to antifungal drugs, the resistance of isolates to fluconazole, ketoconazole and clotrimazole were determined 32%, 28% and 14%, respectively. In disc diffusion, all isolates have an acceptable sensivity at 10 - 20 micro L of the oil and 30 micro L inhibit the growth completely in plate. Minimum Inhibitory Concentrsation [MIC] by microdilution broth method was 500ppm and 750ppm in 72% and 28% of isolates, respectively, and Minimum Fungicidal Concentration[MFC] in 70% of isolates were 750ppm and for the rest of the isolates [30%] were 1000ppm. We conclude that use of this native plant, as an antifungal compound, could act as a treatment of the patients with mucosal candidiasis, beside of other drugs in to the future

Distribution of virulence associated genes in isolated Escherichia coli from avian colibacillosis

Colibacillosis is one of the most prevalent diseases in the world that causes multimillion-dollar annual losses. In order to evaluate molecular epidemiology of some virulence associated factors in Escherichia coli, isolated from poultry, the presence of iut A, iss, hlyF, omp T, iro N, afa, sfa [S] and pap G [II] were investigated by multiplex PCR assay. Two hundred thirty four Escherichia coli isolated from avian colibacillosis [APEC] and fifty four fecal E. coli isolates from the feces of apparently healthy birds [AFEC] were investigated for presence of some virulence associated genes by two panel of multiplex PCR. Statistical analysis was performed using x[2] test, the p-value was

[Effect of Zataria multiflora boiss. essential oil on growth curve and shigatoxin 2 production of enterohemorrhagic Escherichia coli O157:H7]

Zataria multiflora Boiss. is one of the Iranian traditional spices and it has antimicrobial effect on the pathogenic bacteria which are agents for some current foodborne intoxications. The present study was conducted to evaluate the antimicrobial activity of Zataria multiflora Boiss. Essential oil on Escherichia coil 0157:H7. Minimum inhibitory concentration [MIC] and minimum bactericidal concentration [MBC] of Zataria multiflora Boiss. Essential oil [EO] for Escherichia coil 0157:H7 at 35°C, the effect of subinhibitory concentrations of EO on growth curve of bacteria up to 24 hours at 25 and 35°C and also production of shigatoxin 2 [Stx2], at 35°C has been determined. MIC and MBC of EO which have been evaluated were 0.04 and 0.06%, respectively the effect of subinhibitory concentrations of EO on bacterial growth curve during 24 hours has been determined by spectrophotometer device at 25 and 35°C and also production of shigatoxin 2 [Stx2], at 35°C has been determined. Subihibitory concentrations of EO, significantly, decreased the production of Stx2 at 35°C in a dose dependent manner. The results showed that Zataria multiflora Boiss. Essential oil had inhibitory effect on Escherichia coli 0157:H7 and it can be used as a natural preservative in food industry

[Molecular study on Ehrlichia canis in thrombocytopenic dogs]

Infection with Ehrlichia canis, a gram negative obligatory intracellular bacterium, causes canine monocytic ehrlichiosis which is the worldwide disease in dogs. The objective of this study was to investigate the prevalence of E. canis in thrombocytopenic dogs using nested PCR and diagnostic role of thrombocytopenia in the infection. Blood samples collected from 40 dogs attended in Teaching small animal hospital of Tehran University were classified as group A [platelet counts below 101.000/microL, thrombocytopenic, n=11], B [101.000-200.000/microL, thrombocytopenic, n=15] and C [platelet counts more than 201.000/microL, non-thrombocytopenic, n=14] according to their platelet counts. 16S rRNA was analyzed by nested PCR using specific primers. 16S rRNA gene fragment of E. canis were detected in five samples of group A [45.5%], three samples of group B [20%], and one sample of group C [7.1%]. Prevalence rate of infection was statistically higher in group A than the other groups [p=0.02]. In total, approximately one third of thrombocytopenic dogs had demonstrable E. canis infection [30.7%]. While thrombocytopenia cannot be considered as specific marker for detection of E. canis infection, it can be used as a surveillance test prior to other diagnostic methods

[Effect of climatic factors on canine Lime borreliosis in Iranian Caspian sea littoral provinces]

Lyme borreliosis is a worldwide zoonotic disease caused by spirochetes of the Borrelia burgdorferi sensu lato complex. There are no reports on this subject in dogs from Iran. Determining the serologic prevalence level of produced antibodies against Borrelia burgdorferi sensu lato complex in three Caspian littoral provinces of Iran and studying the effect of climatic risk factors on it are the first aims of this study. During the period from July to September 2009 a seroepidemiological study was conducted on 273 dogs in three Caspian provinces of Guilan, Mazandaran and Golestan, Iran's known habitats of tick [Ixodes ricinus]. In order to study the correlation between infection distribution and climatic factors by geographic information system [GIS], geographic position of seronegative and seropositive dogs was overlaid on climatic maps of Guilan, Mazandaran and Golestan provinces. Multivariate regression model and correlation matrix analyses were used for statistical analysis. From 273 serum samples in the whole studied area, 22 [8.1%] showed antibodies against B. burgdorferi sensu lato complex. The seroprevalence of B. burgdorferi sensu lato in provinces of Guilan, Mazandaran and Golestan were 0.0% [0.91], 2.2% [2.91] and 22% [20.91], respectively. Mean annual temperature had positive and significant correlation with B. burgdorferi sensu lato complex seroprevalence in sampled dogs of the three north provinces [p<0.05]. Regarding the seroprevalence of Lyme borreliosis in dogs of three Caspian provinces of Iran, more attention must be paid to this disease, especially in Golestan province. This is the first study on the role of climatic factors in canine Lyme borreliosis in Iran

[Pathotyping of isolated Escherichia coli from domesticated calves and poultry using modern DNAmicroarray technique]

Detection of virulence factors harbored in Escherichia coli strains is important to determine a genotyping profile, and the pathotype of an Escherichia coli isolate. The objective of this study was to determine the pathotype of E. coli strains isolated from calves and poultry domesticated in Iran by using DNA microarray technology. In this study, 67 strains of isolated E. coli from calves and poultry [51 from calves and 16 from poultry respectively] were monitored for the presence of different virulence factors. The pathotype of each strain was made using DNA Microarray technology. The array used 109 probes for the common virulence genes of Escherichia coli and 154 probes for virulence genes that were specifically included pathotypes. Results showed that Escherichia coli strains from calves were 47% EHEC, 15.68% EPEC, 15.68% UPEC, 1.96% ETEC, 1.96% a combination of EPEC and UPEC, and 17.64% of strains non-specific to any of the pathotypes. In the samples from poultry, 62.5% of strains were pathotyped as APEC, 31.25% ExPEC, and 6.25% non-specific to any pathotype. This study revealed that DNA Microarray, as compared with other traditional molecular techniques, is a powerful tool for demonstration of the genotyping profile and pathotype of Escherichia coli strains

Antimicrobial resistance profile and presence of class I integrongs among Salmonella enterica serovars isolated from human clinical specimens in Tehran, Iran

Salmonella is one of the leading causes of food-borne diseases. Increasing occurrence of antimicrobial resistance, especially multidrug-resistance, in Salmonella serovars is a major public health problem worldwide. This study was carried out to detect class I integrons and antibiotic resistance profiles in clinical isolates of Salmonella serovars collected from seven hospitals in Tehran during November 2009 to June 2010. Antibiotic susceptibility profile of 19 antibiotics against 58 Salmonella isolates commonly used in humans was determined using disk diffusion assay. Minimum inhibitory concentration against ceftriaxone and ciprofloxacin was studied. PCR assays were used to detect class I integrons. Among 58 Salmonella isolates, 72.4% were Salmonella enterica serovar Enteritidis, 8.7% were Salmonella enteric serovar Typhimurium and 18.9% were other serovars. Of the total 58 Salmonella serovars, 43 [74.1%] were multidrug resistant and showed resistance to three or more antibiotic families. Class I integrons were identified in 38 [88.3%] MDR Salmonella isolates. Ciprofloxacin minimum inhibitory concentration ranged between 0.125-2 microg/ml for four isolates and other four isolates exhibited resistance to ceftriaxone [MIC 64-256 microg/ml]. The high prevalence of class I integrons was seen in our MDR Salmonella isolates and class I integrons might play an important role in the dissemination of antimicrobial resistance determinants

[Detection of EHXA, STX[1] and STX[2] virulence genes in non-O157 Escherichia coli isolated from cattle and determination of antibiotic resistance profile of these isolates]

Shiga toxigenic Escherichia coli is one the most important bacteria within Bacteriacae. The bacteria infect humans and a wide spectrum of animals, resulting in dangerous consequences such as hemolytic uremic syndrome and hemorrhagic colitis. In the current study, the prevalence of hemolysin [ehxA] and Shiga toxin [stx1 and stx2] virulence genes in non-O157 Escherichia coli, isolated from cattle stool samples, was evaluated by Multiplex PCR. The animals were referred to the Large Animal Teaching Hospital of the Faculty of Veterinary Medicine, University of Tehran. The antibiotic resistance profiles of the isolates were assessed against seven usual antibiotics used in veterinary medicine. In the PCR study of 39 non-O157 Escherichia coli strains isolated from cattle stool samples, 10 samples were found positive for stx1 or stx2 genes. The prevalence of ehxA gene was zero,which is significantly lower than that mentioned in papers reporting on this issue. As expected, the prevalence rate of stx genes in cattle isolates was usual [nearly 25%]. The prevalence of stx2 was greater than the prevalence of stx1. All isolates were multiple resistant to two or more antibiotics, including ampicillin, erythromycin, polymixin-B, tetracycline, trimethoprim-sulfamethoxazole, gentamicin and/or cephalotin

[Virulence genes of verotoxigenic E. coli isolated from raw milk and unpasturized cheese]

Verotoxigenic strains of E. coli mostly contain one or both of stx1 and stx2 genes. Both of these genes play a role in pathogenicity of the bacteria. These strains cause bloody diarrhea, uremic haemolytic syndrome and purpura thrombocytopenia. Because of a high probability of the presence of verotoxigenic strains of E. coli in various foods, especially milk and cheese, and due to the importance of these strains to human health, we aimed to determine the presence of verotoxigenic strains of E. coli in unpasteurized milk and cheese by PCR. In this study, 200 samples of raw milk and 80 samples of unpasteurized cheese were collected, and verotoxigenic E. coli were isolated using selective media. PCR was used to determine some virulence genes including stx1, stx2, eaeA and hlyA. Thirty-eight and 14 E. coli samples were isolated from raw milk and unpasteurized cheese, respectively. The isolates were examined by PCR in order to find the O157:H7 specific DNA and stx1, stx2, eae and hlyA genes. Two out of 38 isolates originating from raw milk were typed as O157:H7, both of them containing stx2, eaeA and hlyA genes. Another isolate, which was not O157:H7, also contained the stx2 gene. No isolates possessed the stx1 gene. None of the isolates originating from unpasteurized cheese samples contained any of the virulence genes

relation of water contamination and colibacillosis occurrence in poultry farms in Qom province of Iran

Seventy poultry farms' drinking water was tested for Escherichia coli contamination in Qom province in Iran. The cases of colibacillosis from positive farms were also collected and tested. The isolates were examined for serotype, detection of virulence genes by multiplex PCR and antibiotic resistance. Thirty poultry farm water samples were E. coli positive [18.57%], although 13 E. coli isolates were recovered from carcasses of related farms. The isolates belonged to O2 serogroup and one O157, with approximately 29% of the strains being non-typeable. Two isolates from water and carcasses were serotyped O2 and one sample serotyped O157, which needs to be further studied. The PCR method was on the basis on showing virulence genes of espB, stxl, stx2 genes were common in a sample from both water and carcass, although five samples from both water and carcass shared a stxl gene as well. All isolates showed maximum sensitivity and resistance to lincospectine and tetracycline, respectively

Evaluation of antibodies levels against Escherichia coli, rotavirus and coronavirus in the colostrum of non-vaccinated cows in southern Tehran, Iran

A competitive enzyme-linked immunosorbent assay [ELISA] kit was used for the evaluation of antibodies against Escherichia coliK99, rotavirus and coronavirus in colostrum samples of 240 non-immunized Holstein dairy cows in southern Tehran, Iran. Antibody levels against E. coli K99, coronavirus and rotavirus were higher than a 20% inhibition threshold in 76%, 99% and 100% of samples, respectively. From a total of 240 samples 14 cases [5.83%], 222 cases [92.5%] and 240 cases [100%] showed the strongest positive results [4[+]] for antibodies against E. coli, coronavirus and rotavirus, respectively. These colostrum samples were considered as high titre colostrum. The results showed that only a small number [5.83%] of colostrum samples had enough antibodies to protect the calves against diarrhea due to E. coli K99 after passive transfer. In the cases of rotavirus and coronavirus it was concluded that the colostrum samples obtained from non-immunized, naturally infected cows contained enough antibodies to develop passive immunity against rotavirus and coronavirus in suckling calves

[Effect of zataria multiflora boiss. essential oil on enterotoxin production by staphylococcus aureus ATCC 6538]

Staphylococcal food poisoning does not result from the ingestion of S. aureus per se, but rather from enterotoxins which are pre-formed within the food. There is no report on effect of Zataria multiflora Boiss. essential oil on enterotoxin production by this microorganism. The aim of this study was to evaluate the effect of different concentrations of Zataria multiflora Boiss. essential oil on enterotoxin production by S. aureus. This study was done as a factorial model using different concentrations of EO [0, 0.005, 0.015 and 0.03%].The bacterial growth was evaluated during 43 days of incubation and enterotoxin production was analyzed using a enterotoxin assay kit. The growth of the organism was completely inhibited by the EO=0.03%. Enterotoxin production was not affected by sub-inhibitory concentration of EO at level of 0.005%, while it was significantly [p < 0.05] inhibited by increasing subinhibitory concentration of EO to 0.015%. The adverse effect of sub-inhibitory concentration of Zataria multiflora Boiss. EO on enterotoxin production by S. aureus, demonstrated in this study, suggests the potential application of this EO as a natural antimicrobial in foods

[Identification of mycobacterium avium subsp paratuberculosis in fecal samples of holstein-friesian cattle using molecular and cultivation methods]

Mycohacterium avium subspecies of Paratuberculosis is the cause of paratuberculosis or Johne's disease. Paratuherculosis is a chronic and progressive infectious enteric disease that affects domestic and wild animals, mostly ruminants. This study was carried out for detection of subclinical forms of Johne's disease using direct PCR and cultivation methods. A total 119 fecal samples were collected Holstein-Friesian cattle herds in Razavi khorasan province of Iran. Among these, 16 samples were taken from cows with advanced clinical signs of Johne's disease. 101 were taken from cows without clinical signs. The fecal samples were tested for presence of Mycobacterium paratuberculosis by directed Nested-PCR and culture on Herrold s media with and without Mycobactin J. The samples of cows with clinical signs, 14[87.5%] and 13[81.3%] were positive by PCR and cultivation assays, respectively. These numberes for the 101 cows without clonical signs of Johne's disease were 10[9.7%] and 12[11.7%]. In conclusion, according to the current report of bacterial detection from different places and the economic importance of John's disease, it is logical and essential that the prevalence rate of the disease in dairy cattle is initially determined by at least two different types of samples and tests and afterwards the control programs are adopted