Identification of Burkholderia mallei Isolates with Polymerase Chain Reaction-Restriction Fragment Length Polymorphism.

Burkholderia mallei is the main cause of glanders as a dangerous contagious zoonosis disease that is mostly observed in single-hoofed animals, especially horses. Modern molecular techniques have been recently employed to improve epidemiology for identifying and searching for strains of this bacterium at different times and locations. Due to the unknown number of circulating strains and lack of preventive methods, glanders is still observed in the form of epidemics. The present study aimed to evaluate six field isolates plus two laboratory strains of Borkolderia mallei and Burkholderia pseudomallei using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. All the isolates and strains were microbially cultured in the glycerol nutrient and glycerol agar media. The individually grown colonies of the bacterium were used in the biochemical tests. The DNA of isolates was extracted by boiling, and the PCR-RFLP test was conducted on their genome. Finally, the bacterium was injected into guinea pigs to induce the Straus reaction. The biochemical assays (or bioassays) confirmed the isolates as Burkholderia mallei. The PCR-RFLP assay demonstrated a product for Burkholderia mallei with a length of 650 bp. Nevertheless, 250 and 400 bp were produced for Burkholderia pseudomallei. The swollen scrotum pointed to the occurrence of the Straus reaction. The PCR-RFLP is a proper differential diagnosis technique for B. mallei; moreover, it is a suitable method for differentiating between Burkholderia mallei and Burkholderia pseudomallei. This technique can detect Burkholderia mallei in a short time with high precision and sensitivity.

Evaluation of Heating and Irradiation Methods for Production of Purified Protein Derivative (PPD) of Mycobacterium Tuberculosis.

Tuberculin skin test, also known as the tuberculin or purified protein derivative (PPD) test, is an extensively applied diagnostic test for the detection of primary infection with Mycobacterium tuberculosis (Mtb). The production of PPD is accompanied by some difficulties that require a series of modifications in the production and purification processes. The present study aimed to determine the facilitation level of the manufacturing process by modifying evaluation methods for the production of PPD tuberculin. Mtb strains were cultured in Lowenstein-Jensen media, and the cultured strains were inoculated into the Dorset-Henley liquid medium by the biphasic medium of potato-Dorset-Henley. After incubation, flasks containing cultured strain were selected for bacterial inactivation, and the optimal gamma radiation dose(s) was determined. Tuberculoproteins were precipitated by ammonium sulfate (AS) and Trichloroacetic acid (TCA). Protein concentration was determined using the Bradford and Kjeldahl protein assay methods. Finally, the lymphocyte transformation test and potency test were performed. Based on the results, the Dorset-Henley liquid medium is suitable for the massive growth of the bacterium. The transferal of Mtb from solid to liquid medium was directly carried out without intermediate culture. It was found that during tuberculoprotein production, heating at 100°C for 3 h would be safe for killing mycobacterium. Furthermore, the simultaneous use of heating and gamma irradiation (8 kGgy) killed all of the mycobacteria, while doses of 1, 1.5, and 7 kGy decreased a significant number of bacterial cells. The results also indicated that the concentration of tuberculoprotein extracted by TCA precipitation method was higher than that obtained by AS precipitation. The tuberculoproteins which were produced by these two methods in the lymphocyte transformation test were not significantly different in terms of potency (P>0.05). Moreover, due to the high volume of produced protein, the protein measurement was more efficiently carried out by the Kjeldahl method, compared to the Bradford method. Finally, the results of the present study demonstrated that in addition to the novel approach of gamma irradiation, optimum methods are efficient and applicable in the production of PPD tuberculin.

Detection and Isolation of Mycoplasma capricolum Subspecies Capricolum from East Azerbaijan Sheep Flocks.

Mycoplasma capricolum subspecies capricolum (Mcc) is one of the causative agents of contagious agalactia (CA), which is an important disease in sheep and goats in the Mediterranean and Middle East countries. Mycoplasma agalactiae is the classic agent of CA in sheep and goats. Mycoplasma mycoides subspecies Capri (Mmc), Mycoplasma capricolum subspecies capricolum (Mcc), and Mycoplasma putrefaciens (Mp) produce a clinically similar disease, more often in goats. The aim of the present study was to detect Mcc in sheep flocks in East Azerbaijan Province of Iran. Milk, ear canal, and eye swab samples were collected from 49 sheep flocks with clinical signs of CA or a history of a disease. All the samples were examined using both culture and molecular methods. In the molecular method,positive samples for the Mycoplasma genus were tested for M. mycoides cluster and Mcc. From 272 samples, 67, 87, and 62 samples were shown to be positive using the culture method, polymerase chain reaction (PCR) method, and both culture and PCR methods, respectively. Mcc was detected in all the four M. mycoides cluster positive samples, including milk, ear canal, and eye swab samples. This is the first report of Mcc detection from East Azerbaijan. Our results showed that eye, milk, and ear canal samples could be suitable sources for Mcc detection in sheep flocks.

Outbreak of avian mycobacteriosis in flocks of domestic pigeons: An epidemiological approach.

BACKGROUND AND OBJECTIVES: Pigeons are extensively kept for homing and racing purposes in Iran. The main objective of this study was to investigate dissemination of M. avium subsp. avium (MAA) in pigeon aviaries in Tabriz, North-western Iran. MATERIALS AND METHODS: Postmortem pathologic specimens from thirty-nine out of 140 birds collected from private flocks (n=3), were subjected to bacterial culture out of which 3-4 mycobacterial isolates were recovered. RESULTS: Applying a five-PCR diagnostic algorithm targeting short but definitive stretches of 16S rRNA and RV0577 genes, IS6110, IS901 and IS1245 genomic loci, proved all the isolates were MAA. They were either IS901+/IS1245+(n=22) or IS901+/IS1245- (n=12). When four healthy cattle sensitized against Mycobacterium bovis AN5 and Mycobacterium 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. CONCLUSION: We believe the extent of such epidemiological impact deserves further investigation if progress in control of bovine tuberculosis is intended.