Nanodiagnostic method for colorimetric detection of Mycobacterium tuberculosis 16S rRNA

A nanodiagnostic method using nucleic acid sequence-based amplification (NASBA) and gold nanoparticle probes (AuNP probes) was developed for colorimetric detection of Mycobacterium tuberculosis. The primers targeting 16S rRNA were used for the amplification of mycobacterial RNA by the isothermal NASBA process. The amplicons were hybridized with specific gold nanoparticle probes. The RNA-DNA hybrids were colorimetrically detected by the accumulation of gold nanoparticles. Using this method, 10 CFU ml-1 of M. tuberculosis was detected within less than 1 h. Results obtained from the clinical specimens showed 94.7% and 96% sensitivity and specificity, respectively. No interference was encountered in the amplification and detection of M. tuberculosis in the presence of non-target bacteria, confirming the specificity of the method. © 2009 Humana Press Inc

A comparison of virulence of intraperitoneal infection of Burkholderia mallei strains in guinea-pigs

Male guinea pigs show high susceptibility to Burkholderia mallei and have been used as animal models in glanders studies. The purpose of our study was to elucidate glanders comparative pathogenesis in guinea pigs. We present here the histological changes and bacterial isolation that develop over time in guinea pigs inoculated intraperitoneally (IP) with two strain of B. mallei. Ten male guinea pigs were inoculated intraperitoneally with either the standard strain of Burkholderia mallei or B. mallei strain from Siberian tiger at the Tehran zoo individually, then euthanized at multiple time points post inoculation. Histopathologic changes were similar in both groups and consisted of pyogranulomatous inflammation. In the standard strain study guinea pigs, changes were first seen at 48 hours in liver and heart then in spleen, lung, and kidney at day 3. These changes generally reached maximal incidence and severity by day 3 but decreased by comparison in all tissues except the liver, lung and kidney. Changes were first seen in Siberian tiger strain study guinea pigs also at 48 hours in lung, liver and spleen. At day 3, changes were present in liver, spleen and mediastinal lymph nodes. These changes were maximal at day 4 and 5. In contrast there are differences in incidence and severity between the two strain study guinea pigs. Our findings based on histopathological study indicate that Siberian tiger strain has more severity in gross and necropsy examination but in pathologic lesion was qualitatively similar generally. Additionally, by bacterial isolation, we confirmed the presence of B. mallei

Avian tuberculosis in flocks of pigeons and its potential impact on tuberculination of cattle

Aims and objectives: The Mycobacterium avium subsp avium (MAA) is a slow-growing, frequently-encountered mycobacterium in the environment that causes tuberculosis mainly in birds and sometimes in farm animals. As a favorite pet bird, the pigeon is extensively kept for homing and/or racing purposes in Iran, therefore, the main objective of this study was to investigate dissemination of M. avium subsp avium (MAA) in pigeon aviaries in Tabriz, North-western Iran. Methods: From a total of 140 birds collected from private flocks (n = 3), The pathologically changed lungs and the lymph node were examined histologically by staining with Ziehl–Neelsen (Z–N) and hematoxylin–eosin; 39 were subjected to bacterial culture, out of which 34 mycobacterial isolates were recovered. Mycobacterial DNA was isolated according to the previously described method (Van Soolingen et al., 1993). Applying a five-PCR diagnostic algorithm targeting short, but definitive stretches of 16S rRNA and RV0577 genes, IS6110, IS901 and IS1245 genomic loci, all the isolates were identified as MAA. PvuII-IS901 RFLP typing was also performed on all isolates. Results: They were either IS901+/IS1245+ (n = 22) or IS901+/IS1245 (n = 12). In IS901-RFLP strain typing of a subset of the isolates (n = 22), they were classified into five distinct multi-banded but similar patterns, namely PA (n = 13), PB (n = 5), PC (n = 2), PD (n = 1) and PE (n = 1). No correlation between IS901-RFLP genotype and presence/lack of IS1245 was noted as isolates both holding and lacking IS1245 were found to share PA and PB genotypes. Whilst no case of mixed infection with more than one strain was detected in any single bird, it was not possible to extend this observation to the aviary level as original colonies of birds were not recorded. When four healthy cattle sensitized against Mycobacterium bovis AN5 and M. avium D4 were tuberculinated, the results confirmed the observed skin reactions against bovine tuberculin in animals sensitized with M. avium were large enough to complicate test interpretation. Conclusions: It is believed that the extent of such epidemiological impact deserves further investigation if progress in the control of bovine tuberculosis is intended. This indicates the importance of identification of the causative agent before any conclusions are made, based solely upon the results of the skin test and histopathological examination

Genomic pattern analysis of burkholderia mallei field isolates by pulsed-field gel electrophoresis (Pfge) discriminatory typing

Background and Objectives: Glanders is a serious zoonotic disease caused by Burkholderia mallei. Prevention, control, and treatment strategies of glanders are prerequisites for microbial source tracking. The present study was aimed to analyze the genomic pattern of B. mallei Iranian field isolates by pulsed-field gel electrophoresis (PFGE) typing. Materials and Methods: B. mallei isolates were aerobically cultured in nutrient broth/agar supplemented with glycerol 4 for 48 h at 37°C. API 20NE identification system was used for the biochemical characterization. Genomic DNA of bacterial isolates was extracted using OIE-recommended protocol. Molecular identification of bacterial isolates was done based on amplification of BimA and IS407-flip genes. PFGE was applied to prepare the genomic pattern of B. mallei isolates. The guinea pig was used as a suitable model for studying the histopathological characterization of B. mallei. Results: In both enzymatic digestion patterns by using Af1II and VspI, we found three different clonal types; �) PFGE type of B. mallei Razi 325 strain, ��) PFGE type of Tiger, Kordan, and Oshnavieh strains, and ���) PFGE type of Semirom strain. B. mallei Razi 325 was categorized as unrelated strain which was belonged to the different cluster differing more than four bands. Conclusion: PFGE showed more discriminatory power and considerable reproducibility for molecular typing of B. mallei strains in our study. It is standardized the approaches for outbreak detection, pathogen phylogeny, molecular epidemiology, and population studies. © 2021 The Authors. Published by Tehran University of Medical Sciences

An OIE-Approved Reference Laboratory for Veterinary Mycobacteriology in Iran

BodyMycobacterial diseases are an important group of animal infections, which have tremendous economic and public health ramifications. Bovine tuberculosis and paratuberculosis, both identified years ago, have no effective vaccine or treatment controls in farm animals. In Asia, there are only seven countries where test-and-slaughter schemes are currently ongoing to control bovine tuberculosis. On the other hand, among five mycobacteriology reference laboratories, licensed by OIE, in the world, none is located in Asia. Iran has proved to have an efficient control program against tuberculosis in cattle since the 1960s. While the locally made PPD tuberculins required for the testing of cattle are apparently useful in the Iranian scheme, there is no organised establishment in Iran to support the control programme with bacteriological services. This presentation considers the needs and potentials of a reference laboratory facility in Iran