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Identification of Mycobacterium Tuberculosis Complex, Using Molecular Methods
M Eramabadi, K Tadayon, N Mosavari, R Keshavarz, R Banihashemi, R Ghaderi, M Sekhavati, M Ahmadi, P Eramabadi, E Khodaverdi Daryan,
Volume 7, Issue 5 (supplement Issue( Bacteriology)[PERSIAN] 2014)
Abstract
Abstract Background and Objective: A high level of homogeneity observed within all bacteria in the Mycobacterium tuberculosis complex makes a property that seriously challenges traditional biochemical-based identification methods of these pathogens in the laboratory. The work presented here was conducted to characterize Mycobacterium tuberculosis complex isolates in Golestan, Northern Iran. Material and Methods: Between 2008 and 2010, 42 mycobacterial isolates were collected from clinical tuberculosis-suspected patients in Golestan province. The isolates were sub-cultured on fresh Mycobacterium-specific culture media including glycerinated and pyruvated Lowenstein-Jensen slopes. The isolates were subsequently subjected to a PCR-based identification scheme coined Huard-Warren method. This strategy consisted of three individual algorithms namely, 16SrRNA RV typing (Rv0577, Rv3877.8, Rv1970, Rv3120, Rv1510 and IS1561) and RD typing (RD1, RD 4, RD9 and RD12). Results: All isolates were proved to be M. tuberculosis. Furthermore, none of the patients were being infected with any other member of the M. tuberculosis complex or simultaneously co-infected with two mycobacteria. This fundamental observation was independently obtained by specific culture media, RV typing and also RD typing. Conclusion: Considering the fact that cattle and sheep farming play an important role in the economy of the region, absence of Mycobacterium bovis in the studied isolates can be unexpected to some extent. Huard-Warren which is a simple and cost-effective identification method can be used in both reference and regional laboratory for differential diagnosis of tuberculosis. Keywords: Mycobacterium Tuberculosis Complex, Huard-WarrenMethod, 16SrRNA, Golestan Province, RD Typing, RV Typing
A Simplified Van Erth Single Nucleotide Polymorphism (SNP) Typing Method of Bacillus Anthracis Applicable by Traditional Thermocycler Machines
Najafi Olya, Z. (bsc), Tadayon, K. (phd), Ghaderi, R. (bsc),
Volume 9, Issue 1 (March, April[PERSIAN] 2015)
Abstract
Abstract SNP typing is now a well-established genotyping system in Bacillus anthracis studies. In the original standard method of Van Erth, SNPs at 13 loci of the B. anthracis genome were analyzed. In order to simplify and make appropriate this expensive method to low-budget laboratory settings, 13 primer pairs targeting the 13 corresponding SNPs were designed. Besides, a universal PCR protocol was developed to enable simultaneous amplification of all loci by conventional PCR machines. The efficiency of this approach was approved by applying on nine isolates of B. anthracis. We recommend using this modified procedure as an efficient alternative to Van Erth method until developing newer and affordable techniques. Keywords: Bacillus Anthracis, Genotyping, SNPs, PCR
Genomic Characterization of the Vaccinal Strain of Mycobacterium Avium Subspecies Paratuberculosis (MAP) 316F by MIRU-VNTR
Zahra Ebrahim , Keyvan Tadayon , Nader Mosavari ,
Volume 9, Issue 4 (sep,Oct 2015 2015)
Abstract

Abstract

       Background and Objective: Paratuberculosis is a chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). this study aimed to characterize the genome of the MAP 316F strain.

      Methods: The MAP 316F strain was subjected to the PCR-F57 and PCR-IS900 experiments in order to ensure its identity as MAP. This was followed by application of the Thibault genotyping system consisting of eight loci including 292, x3, 25, 47, 3, 7, 10 and 32. Required genomic material for all experiments was prepared using the simple method of boiling. Gel electrophoresis findings related to the typing PCRs were backed by sequencing of amplification products.

      Results: In PCR amplification, eight products with the size of 300, 298, 350, 217, 208, 203, 803 and 649 bp were detected at 292, X3, 25, 47, 3, 7, 10 and 32 loci, holding 3, 2, 3, 3, 2, 2, 2 and 8 copies of TRs at these loci, respectively.

      Conclusion: This genomic pattern is matched with that of the MAP 316F vaccine strain from the French Merial company and also the MAP K10 fully-sequenced strain.

       Keywords: Mycobacterium avium subsp. paratuberculosis, Genomics, Genotyping techniques, Strain


Genetic Structure of SSR1 & SSR2 loci from Iranian Mycobacterium Avium Subspecies Paratuberculosis Isolates by a Short Sequence Repeat Analysis Approach
Aida Chalesh , Keyvan Tadayon ,
Volume 9, Issue 5 (Nov,Dec-2015 2015)
Abstract

Abstract

        Background and Objective: Paratuberculosis has been repeatedly reported from Iranian ruminant herds. The extrem fastidious nature of Mycobacterium avium subspecies paratuberculsos hinders genomic diversity studies of the pathogen. Short Sequence Repeat analysis is one of the genome-based approches recently developed to overcome this difficulty. In this study we describe the application of SSR genotyping on three Iranian MAP type strains plus the III & V vaccinal strain.

        Methods: All the bacteria were examined by PCR-F57 and PCR-IS900 experiments in order to authenticate their identity as MAP. SSR genotyping using SSR1 & SSR2 loci was conducted according to the Amonsin method. PCR amplicons were sequenced to guarantee the accuracy of findings.

        Results: At SSR1 locus two allels were identified, a larger allel of 770 bp and a smaller allel of 763 bp long. At SSR2 only a single allele, 800 bp long, was detected. Two Iranian bovine and ovine MAP isolates along with the vaccinal III & V strain shared a single SSR1/SSR2 pattern while a different SSR1/SSR2 was represented by the third (caprine) Iranian MAP isolate.

        Conclusion: While finding a shared SSR type between the two Iranian MAP isolates and the III & V strain might represent a mutual ancestral background but this has to be assessed through further studies. Detection of two SSR genotypes between three Iranian type strains is likely a reflection of more MAP clones in Iran.

       Keywords: Mycobacterium avium subspecies paratuberculosis (MAP), SSR genotyping, Genetic marker, Genetic locus


In Vitro Protoscolicidal Activity of Pomegranate (Punica Granatum) Rind and Barberry (Berberis Vulgaris) Alcoholic Extracts against Hydatid Cysts Caused by Echinococcus granulosus
Shahab Shiri Hamedani, Mohsen Mansouri, Sina Shiri Hamedani, Parham Tadayon, Peyman Aslani, Mohammad Mohsen Homayouni,
Volume 16, Issue 4 (Jul-Aug 2022)
Abstract
Background and objectives: Echinococcosis is a global cosmopolitan zoonotic disease and a major veterinary and public health issue. In humans, echinococcosis usually develops following close contact with infected dogs or ingestion of the parasite eggs. Until now, no effective vaccine has been commercially developed, and treatment is only focused on controlling hydatidosis. This study was conducted to evaluate the protoscolicidal activity of alcoholic extracts of pomegranate rind and barberry.
Methods: The alcoholic extracts of pomegranate rind and barberry were prepared by mixing 330 g of powdered plants with 1,000 ml of 70% ethanol. A concentrate of viable protoscolices (PCSs) was obtained from hydatid cysts found in the lungs and liver of sheep. Next, PCSs were treated with four different concentrations (5, 10, 20, 30, and mg/ml) of each extract for 10, 20, 30, and 60 minutes. The eosin exclusion test was performed to assess viability of the PCSs.
Results: The mortality rate caused by treatment with the extracts ranged between 25% and 100%. Complete inactivation of PCSs was achieved after 60 minutes of exposure to 15 mg/ml of the pomegranate rind extract and 30 mg/ml of the barberry extract.
Conclusion: Given their favorable anti-PCSs activity, combination of conventional synthetic albendazole with the alcoholic extracts of pomegranate rind and barberry might induce higher anti-PCS activity with lower side effects. It is recommended to evaluate the anti-PCSs activities of the pomegranate rind and barberry alcoholic extracts in vivo and ex vivo.
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