[Immunogenic evaluation of recombinant chimeric protein containing EspA -Stx2b- Intimin against E.coli O157 H7]

Background and Aim: Enterohemorrhagic Escherichia coli [EHEC] cause a wide spectrum of infections, such as diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome. Considering the risks associated with antibiotic therapy against EHEC infection, vaccines can be a promising method for prevention of infections. Recombinant chimeric proteins containing multiple immunogens could induce immunity against bacterial infections. The aim of this study was to evaluate immunogenecity of trivalent chimeric antigen EspA -Stx2b - Intimin against E.coli O157 H7 infection

Material and Methods: In this descriptive-laboratory study, recombinant chimeric protein was expressed in E.coli BL21 DE3 by use of IPTG. The protein expression was evaluated by SDS-PAGE and western blotting analysis. The recombinant protein was purified using Ni- NTA affinity chromatography. The immunization was conducted in mice with purified protein and antibody titers were determined by ELISA. Following immunization, mice were infected with E.coli O157:H7 and evaluated for bacterial shedding and mortality. Using SPSS software, statistical analysis was performed by Duncan's test and T-test

Results: The protein was expressed in E.coli BL21 [DE3] and SDS-PAGE analysis showed expression of recombinant protein with molecular weight of 63kD. Western blot analysis confirmed presence of chimeric protein. ELISA results showed that immunogenic properties of chimeric protein induced humoral response to EspA, intimin and Stx2b. Bacterial shedding in immunized mice decreased to 102 cfu/ml and mortality rate was reduced to 60%

Conclusion: The results showed that the chimeric protein induced humoral response and protected the mice against E.coli O157:H7

[Identification of Campylobacter spp. from poultry skin using methods based on bacterial culture and polymerase chain reactions]

Background: food infections caused by Campylobacter are one of the gastrointestinal inflammations in humans is health and economic losses in the community is important

Objectives: to determine the prevalence of Campylobacter contamination in chicken skin samples of Urmia, using bacterial culture and polymerase chain reactions

Methods: 80 samples of chicken skin from the Protein Gostare Sina slaughter house located in the city of Urmia in equal numbers in the winter and spring seasons were collected. The survival of Campylobacter after 24 hours in refrigerated conditions was studied in samples. Positive samples were used for DNA extraction and PCR. To investigate the phylogenetic isolates, positive samples PCR were sequenced

Results: 58/75% of chicken skin using bacterial cultures, Campylobacter were positive. The Results study the survival Campylobacter in cold conditions after 24 hours, showed that no significant decrease in the survival Campylobacter as well as contamination levels were significantly higher in spring than in winter, which may be due to the high temperature of environment that created the favorable conditions for Campylobacter

Conclusions: chicken skin is the reservoir of Campylobacter. This issue of public health care and control at all stages of production and supply of poultry products, also the transfer of it to other parts of poultry carcasses should be considered

Immunoasssay chromatographic antigen test for rapid diagnosis of Group A beta hemolytic Streptococcus pharyngitis in children: a cross/ sectional study

Group A beta-hemolytic streptococcus [GABHS] is an important pharyngotonsillitis etiologic agent in children. The objective of this study was diagnosis of streptococcal pharyngitis based on rapid antigen detection test and conventional pharyngeal culture. The rapid GABHS antigen detection test was compared to culture on blood agar, the gold standard for the diagnosis of this etiologic agent. Streptococcal antigen was detected in pharyngeal specimens of 34.5% of cases by rapid strip test. We detected group A Streptococcus in 17.2% of pharyngeal culture. There was no agreement between two methods [PV < 0.1]. The negative pharyngeal culture results are probably due to antibiotic usage in 43.2% of patients. Positive rapid test results in pharyngeal swab was age dependent [P < 0.05]. There was good correlation between observing the [petechia in pharynx of patients] and positive rapid test in pharyngeal swab [P < 0.004]. Throat culture results were relatated to previous antibiotic usage [P < 0.03]. The rapid test in pharyngeal swab is helpful for rapid diagnosis and treatment of GABHS pharyngitis. Diagnosis of GABHS pharyngitis based on soley clinical findings is misleading in the majority of cases. Petechia observed in pharynx of the cases was highly predictive of streptococcal pharyngitis

[Shigella dysentery stxA mutant [R170L-A231D-G234E] gene design and optimization of recombinant protein expression and purification]

Background and aims: Shigella dysentery is one of the most important human pathogenic intestinal bacteria. Entrance of the shigella toxin into the epithelial cells inhibits protein synthesis leading to cell death. In spite of great investigations on vaccine production against S. dysentery, studying to achieve significant stxA recombinant protein still remains important. The objective of this study was to determine the appropriate mutation loci and designing stxA subunit synthetic gene, its further expression and optimization and ultimately assaying purification method for further immunization studies

Methods: Three mutant stxA gene including [R170L-A231D-G234E] were designed and the synthetic gene in pET28a plasmid was obtained and confirmed by PCR. Thereafter the plasmid was transformed into the host cell E.coli BL21 DE3 after which gene expression was optimized and protein purity assay was then performed

Results: Preliminary studies led to mutant stop gene design, after which it was confirmed by synthetic plasmid and PCR. Expression and optimization were then performed which resulted in large amount of protein inclusion bodies. Purification of inclusion bodies and protein which resulted solubilization was done with a combinatorial method

Conclusion: With regard to the mechanism of shiga toxin effect and favorable mutation design with new arrangement, less toxicity of expressing protein is predicted than previous other mutants, posing a better vaccine candidate

[Evaluation of the effect of intra cerebroventricular administration of CCPA on reduction of tolerance to the analgesic effect of morphine in rat]

Continuous or long term use of opiate drugs may cause tolerance to the analgesic effect of these drugs, which limits the therapeutic efficacy of these drugs. In this study we evaluated the effects of central administration of 2-chloro-N6-cyclopentyladenosine [CCPA], a selective A1 receptor agonist, on morphine-induced tolerance in rats. Different groups of rats received daily intracerebroventricular [ICV] morphine [10 mg/kg, ip] in combination with saline 5 micro l/rat, or intracerebroventricular [icv] CCPA [20, 40, 80 micro g/5 micro l/rat]. Nociception was assessed by use of a hotplate apparatus [55 +/- 0.5°C]. Using a hot-plate device, the pain inducing effect was recorded when the rat licked its hind paw or jumped. Results showed that different doses of CCPA [20, 40, 80 micro g/5 micro l/rat, icv] delayed the tolerance to the analgesic effect of morphine for 4, 8, and 10 days respectively. In addition, our results showed that CCPA increased the total analgesic effect of morphine. We found that intracerebroventricular administration of CCPA, A1 selective agonists, prevented morphine-induced tolerance to the analgesic effect in rat

Comparison of rectal dose in different techniques of prostate cancer external-beam radiotherapy based on TLD and XR type T GAFCHROMIC[R] film dosimetry

Radiotherapy of prostate carcinoma often results in high doses to surrounding structures, such as the rectum and bladder. Therefore, these organs should be closely monitored. The aim of this study is to evaluate the dose received by the target volume and rectum to compare two different methods of dose measurement with each other and to check the homogeneity of dose in the tumor volume. The dose distribution throughout a planned target volume and the rectum [OaR] in a phantom exposed to 9 MV photon beam, similar to treatment conditions were studied. Several techniques of external beam radiation therapy such as two-, three- and four-field have been planned. Dosimetry was performed using GAFCHROMIC film and TLD-100 chips. The rectal and cancer volume measured doses in treatment were similar to the prescribed doses. The results of two dosimetry types were compared with each other as well as with treatment planning. Rectal dose in three- and four-field [equal tumor dose and equal applied dose] techniques were respectively 23.15, 28.87 and 15.22% lower than the tumor dose. There was not a statistically significant difference between received and prescribed doses. So, this study showed that the Gafchromic film dosimetry can be used for fast dosimetric evaluations

Determining rectal dose through cervical cancer radiotherapy by 9 MV photon beam using TLD and XR type T GAFCHROMIC[R] Film

The goals of the present research were to investigate the rectal dose during four chosen techniques of cervical cancer radiotherapy and to examine how accurately the treatment planning represents dose measurements, and it's practicality for routine use as well as, to determine the homogeneity of dose in tumor volume. The study was carried out using a Nepton 10-PC unit and a Rando phantom. The equipments which were used for dose determination were a Radiochromic densitometer with GAFCHROMIC[R] film [XR type T], and a thermo-luminescent dosimeter [TLD] reader system with TLD chips for rectal and target volume dose determination. Several techniques of external beam radiation therapy such as two-field [AP-PA], three-field and four-field with equal tumor dose and with equal applied dose were planned. The maximum dose received by rectum was caused by two-field technique. The results of two dosimetry types were compared with each other as well as with the treatment planning, however, no statistically significant difference was observed between them [p > 0.05]. In three-field, four -field with equal tumor dose and four-field with equal applied dose, rectal dose was lower, respectively 26.17%, 33.75% and 16.47%, than tumor dose. This study showed that dosimetry using TLD and film during radiotherapy could have a useful role as a predictor of choosing appropriate technique for preventing future rectal complications. Dose limitation to the rectum could possibly be achieved by using three-field and four-field techniques with equal tumor dose while maintaining a high dose to the tumor