We have identified a globally important clonal complex of M. bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of Mycobacterium bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu,1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than

BackgroundInflammatory bowel disease (IBD) is described as a relapsing condition with high morbidity and uncertain complex pathogenesis. The association of Mycobacterium avium ssp. paratuberculosis (MAP) with Crohn’s disease (CD) in human has been debated for decades, however there is no confirmed data to verify such relations in Iran. The aim of this study was to investigate risk factors and a possible role of MAP in Iranian patients with CD.MethodsAnti-MAP antibodies were detected in serum of IBD patients and subjects without IBD (nIBD) through ELISA; MAP DNA and viable MAP cells were identified in patients’ biopsies through nested PCR and direct culture methods, respectively. Principal component analysis (PCA) was used to investigate the risk factors in relation to IBD and MAP infection.ResultsPositivity for IS900 PCR was detected in 64% (n = 18) of CD, 33% (n = 10) of ulcerative colitis (UC) and 9.7% (n = 6) of nIBD samples. Live MAP cells were isolated from biopsies of 2 CD patients only. Among 28 patients with CD, 46% (n = 13) and 39% (n = 11) were positive for antibodies against MAP3865c133–141 and MAP3865c125–133 peptides, respectively, whereas much lower seroreactivity was detected in UC subjects accounting for 3% (n = 1) for MAP3865c133–141 and 16.7% (n = 5) for MAP3865c125–133. A high immune reactivity to MAP epitopes among CD patients was positively correlated with consumption of fast food meals and IBD familiarity. For both CD and UC, breastfeeding period and consumption of fruit/vegetables presented negative correlation with the presence of anti-MAP antibodies.ConclusionsThis study provided evidences that high prevalence of MAP DNA and anti-MAP antibodies in CD patients is significantly associated with the development of CD. Despite the role of several factors contributing to IBD, the presence of MAP DNA and anti-MAP antibodies in Iranian CD patients highlights a possible transmission of MAP from animal-derived products to humans.

To identify strains of Mycobacterium bovis circulating in Iran, we used region of difference, spoligotypes, and variable number tandem repeats to genotype 132 M. bovis isolates from Holstein Friesian cattle. Despite wide geographic origins, the strains were genetically homogeneous. Increased distribution of cattle herds and inadequate control measures may have contributed to strain dispersion. E stimates suggest that globally >50 million cattle are infected with Mycobacterium bovis, causing an annual loss of ≈$3 billion US (1). In Asia, 94% of the 460-million cattle herd (33% of the world's cattle) are in areas with either no or only partial tuberculosis (TB) control programs (2). In 2006, the prevalence of bovine TB in Iran was 0.12% (Iranian Veterinary Organisation [IVO], unpub. data), yet few studies have been conducted on M. bovis in Iran (3-5). To identify the strains of M. bovis in Iran, we used region of difference (RD) typing, spoligotyping, and variable number tandem repeats (VNTR) typing. The StudyFrom 1996 through 2003, we collected necropsy specimens from TB-test reactor cattle from abattoirs in 21 of the 28 Iranian provinces where bovine TB has been reported. Specimens were all respiratory and gastrointestinal lymph nodes and any lungs, spleens, or livers that were visibly affected. All specimens were cultured for M. tuberculosis complex bacteria and incubated for >10 weeks. Of the 470 animals tested, results were positive for 216; however, because of delays in exporting samples to the United Kingdom, only 132 samples contained reculturable isolates with suffi cient growth for DNA extraction. Molecular speciation was determined by RD-PCR (RD1, RD5, RD9, RD10, and RD11) (6). Spoligotyping was conducted according to the method of Kamerbeek et al. RD-PCR showed that all 132 isolates were M. bovis. Spoligotyping identifi ed 8 types (Figure 1). SB0120 was the most common, and 5 others (SB1167-SB1171) were novel patterns and, thus, were specifi c to Iran. VNTR typing identifi ed 23 profi les (Figure 2). ConclusionsRD typing of the 132 isolates confi rmed that they were all wild type M. bovis; none were the M. bovis BCG vaccine strain because they carried the RD1 region. This fi nding is noteworthy because unauthorized vaccination of cattle with BCG has been reported in Iran (IVO, unpub. data). Although previous studies in Iran have reported the isolation of M. tuberculosis from tuberculin-positive cattle (3), our RD9 and RD10 analyses indicated that no isolates were M. tuberculosis. This fi nding suggests that M. tuberculosis is unlikely to be abundant, if even present, on cattle farms of Iran. Similarly, RD5 and RD11 analyses indicated that no isolates were M. africanum or M. microti.The spoligotypes were either identical to the BCG-like (SB0120) pattern (41% of isolates) or were simple variants of it by the deletion of 1 or occasionally 2 single or contiguous blocks of spacers (Figure 1). Because spoligotype changes have been attributed solely to the deletion of spacer units, the BCG-like strains here ...

Introduction: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis or Johne's disease in ruminants. Its role in triggering autoimmunity, including type 1 diabetes mellitus (T1DM), has been reported in recent years. Due to the high contamination rate of MAP in Iran's livestock and the increasing outbreak of T1DM, we investigated this association in a small group of patients with T1DM in Iran. Methodology: Blood samples of 29 T1DM patients and 29 healthy control subjects were tested through enzyme-linked immunosorbent assay (ELISA) to detect antibodies against MAP3865c and ZnT8 homologous epitopes and the presence of MAP DNA. Blood samples were also cultured in mycobacterial growth indicator tubes and Herrold's egg yolk medium containing mycobactin J. Results: The results of ELISA showed a significant difference between T1DM patients and healthy group. IS900 was also detected in 51.72% of T1DM patients but in none of the control group. None of the samples grew in culture media. Conclusions: Due to the presence of MAP DNA and antibodies against MAP peptides in a significant number of T1DM patients compared with healthy control subjects, we may consider MAP as a possible trigger of T1DM in Iran. This indicates that exposure to MAP occurred in the positive subjects. Identifying the sources of contamination and routes of MAP transmission to humans seems necessary to prevent and reduce the burden of MAP infection in Iran.

Human tuberculosis caused by Mycobacterium tuberculosis (Mtb) remains a significant disease in many countries. According to Iran's borders with Afghanistan and Pakistan, which are among the 22 high burden countries around the world, this study was conducted to analyze the current molecular epidemiology of tuberculosis and survey genetic diversity of Mtb strains in Markazi Province in center of Iran. In this experimental study, 75 sputum specimens and one gastric lavage from all smear-positive TB patients admitted to the public hospitals across the Markazi Province were cultured on specific mycobacterial culture media. Genomic DNA was digested by PvuII and transferred to positively charged nylon membrane by southern blotting method and hybridization by PGRS and DR probes. Genotyping of the isolates by PGRS-RFLP and DR-RFLP displayed a wide range of genetic diversity as 25 and 26 genotypes were identified, respectively. Generally speaking, despite the relatively limited number of isolates in the study, high age of patients and also large heterogeneity found in the setting are both in opposition to active circulation of Mtb strains between patients under study either Iranian or Afghan nationals. Thus, it seems that reactivation of latent infection has had the main role in the spread of tuberculosis.

Background: Molecular typing techniques are reliable tools for epidemiological study of tuberculosis because of their power in detecting recent transmission and differentiating reinfection and relapses. Objectives: The present study investigated epidemiological diversity among Mycobacterium tuberculosis strains circulating in three Khorasan provinces, Iran, using 12-loci MIRU-VNTR and spoligotyping. Methods: This study was performed on 140 M. tuberculosis strains selected from the sputum of new cases of pulmonary tuberculosis patients in three Khorasan provinces, Iran. 12 loci MIRU-VNTR and Spoligotyping were performed on all isolates. Results: By MIRU-VNTR analysis, 76 distinct patterns comprising 19 clusters and 57 unique patterns were identified. Based on the results, MIRU10, MIRU26, and ETRF were highly discriminative, ETRD was poorly discriminative and other loci were designated as moderately discriminative. Spoligotyping of isolates revealed 51 distinct patterns: 26 patterns containing 33 strains (23.6%) corresponding to orphan strains and 14 patterns containing 107 strains (76.4%) corresponding to shared-types in the SITVIT2 database. Totally, 103 isolates (73.6%) were classified into 14 clusters containing 2 -56 isolates; the remaining 37 isolates (26.4%) were unique patterns. By combining two techniques, 94 distinct patterns (15 clusters) contained 61 isolates (43.6%), and 79 unique patterns were identified. The discriminatory power (HGDI) of combination of two techniques was 0.962, which was higher than that of each technique alone. Based on the trees designed by Bionumerics software, we differentiated isolates with similar genetic patterns and grouped them together. Two great clusters were Haarlem and CAS lineage. All strains with combined drug resistance related to Beijing strains. Also, all mono drug-resistant strains related to Haarlem family; other strains were susceptible to the first-line antituberculosis drugs. Also, homoplasy was observed in a number of patterns. Conclusions: In MIRU-VNTR typing method, according to the genotype of each area, the loci with high discriminatory power (such as miru10, miru26, and ETRF in Iran) are recommended to be used and the loci with poor discrimination (such as ETRD in Iran) are not.

Although Ag NPs exhibited low cytotoxicity, they were unable to inhibit Mtb growth in vitro. ZnO NPs exhibited strong anti-Mtb activity and inhibited bacterial growth, but exhibited high cytotoxicity to human macrophage cells. By mixing Ag and ZnO NPs at a ratio of 8/2, we acquired a mixture that exhibited potent antibacterial activity against Mtb and no cytotoxic effects on THP-1 cells, resulting in inhibition of both in vitro and ex vivo Mtb growth Figs. 1-3, Tables 1-3.

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