Prevalence of Cep gene of Brucella isolated from clinical specimens in hospitals from Kermanshah of Iran.
MATERIALS AND METHODS
Bacterial strains and verifying of clinical samples
Of 100 serum specimens from patients referred to hospitals of Kermanshah province in 2014 year were collected. These specimens were positive for Wright test (A1/80) and as well as were confirmed by culture and biochemical tests. The average ages of patients were 70-18 years. Then clinical specimens were transferred to the laboratory and stored at -20[degrees]C until use.
Performance of PCR test
Firstly, cep sequence was attained from gene bank and the sequence was assessed to characterize and verifying the complete similarity (Alignment). The primers design for cep gene was carried out by proper softwares. Primers sequences are: F cep: CACGAGGATAGCGGGAATTA, R cep: TTTCGTCAGTGCCCTTCTCT. The condition for PCR in our study include: initial denaturation: 95[degrees] C for 5 min, denaturation at 90[degrees]C for 1 min, annealing temperature; 58[degrees]C for 1 min, extension step: 72[degrees]C for 1 min, 35 cycles and final extension: 72[degrees]C in 5 min. For extraction of DNA from specimens, the kit from Cinnagen Company was applied. After that, the electrophoresis was done for assessing PCR results, then results analyzed by SPSS software.
The results of cep gene from clinical isolates of Brucella
PCR reaction was performed for survey the presence of the pertinent gene in 100 specimens isolated from hospitals of Kermanshah, Iran. Among 100 clinical isolates of Brucella, 54 (54%) had cep gene (Table 1, Figure1). 25 (25%) and 75 cases (75%) of patients were males and females, respectively.
In our study, it was found that 75% of patients were males and only 25% of them were females, which can be due to considering this disease as an occupational disease. Because more men have jobs such as livestock, butchering, agriculture and veterinary medicine are more infected with brucellosis and nearby 90% of veterinary surgeons in the Middle West of the United States obtain this disease (14). Brucella infects phagocyte cells such as macrophages, neutrophils and epithelial cells. Brucella after enter to the cell; locate in vacuole-like structures. These vacuoles are then merging with lysosomes, due to the merge and acidification of the vacuole environment, bacterium excrete some virulence factors. Next, the vacuole membrane will connect to the endoplasmic reticulum, and provides the possibility of intracellular growth of Brucella and cause chronic infection (15). Capsule is one of the most important factors in this bacterium. Capsule has several roles, including anti-phagocytosis, preventing the arrival of antibiotics, preventing dehydration, and antigen binding properties. But the most important role of capsule is anti-phagocytic property (16). In the past it was believed that Brucella didn't have capsule, but recent studies have shown that some species of this bacterium have capsule which containing polysaccharides and proteins. The gene encoding the capsule is named cep and also in Brucella present antigen L which called antigen Vi in Salmonella typhi that has a similar role (17).
The innovation of this study is that the cep gene frequency in serum specimens examined that is very important because presence of cep gene in serum specimens can be indicating of bacteremia and septicemia. Diagnostic value of cep gene from other factors (18) that used for detection by PCR is not less and have a sensitivity approximately them, and because there is not in all strains of Brucella the factors and mechanisms of its pathogenesis is different from other factors, can be used as a complementary diagnostic factor in companying with other factors. Microcapsule also plays an important role in the escape of bacterium from phagolysozyme system. And because the study about distribution of capsule and cep gene was not available, this study has provided the first report on the Brucella status in Kermanshah province. Brucella strains which have eligible microcapsules are more likely to pathogenesis. The microcapsules also play a role in the process of anti-phagocytic and escape from the phagolysozyme system, so the presence of capsule can increase the pathogenicity of Brucella, Accordingly, in incidence of chronic infection is more effective and detecting of chronic infection in the laboratory is difficult (16). Hence can introduce the cep gene as a good candidate for vaccine design also as a promising candidate in detection of this bacterium. So, development and production of drugs which capable of inhibiting cep gene is most importance. In this study, frequency of cep gene was about 54% that shows about 50% of the strains studied in this investigation have high virulence.
According to the first time in this study was revealed the cep gene for diagnosis of brucellosis in the serum. So, cep gene can be used as a diagnostic component with other factors; however; this finding should be studied in animal models and diagnostic and therapeutic strategies linked with this gene will be also investigated.
We thanks to BMSU University for providing facilities and support.
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Rahim Sorouri , Shoaib Khodamoradi , Azad Khaledi  and Davoud Esmaeili  *
 Applied Microbiology Research Center, and Microbiology Department, Baqiyatallah University of Medical Sciences, Tehran, Iran.
 Antimicrobial Resistance Research Center, Avicenna Research Institute, Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
(Received: 17 February 2016; accepted: 10 April 2016)
* To whom all correspondence should be addressed.
Tel: 098-22289941; Fax: 098-21-26127258; E-mail: firstname.lastname@example.org
Caption: Fig. 1 Results of products electrophoresis with cep primers. Wells 3, 5,7. Negative control, wells 2, 4, 6. Examples of positive samples for cep gene and well 8 is related to DNA marker
Table 1. Frequency of cep gene in clinical specimens Variable Positive cases % Total cep gene 54 54 100
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|Author:||Sorouri, Rahim; Khodamoradi, Shoaib; Khaledi, Azad; Esmaeili, Davoud|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Sep 1, 2016|
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