Implementation of a quantitative real-time PCR assay for the detection of Mycobacterium immunogenum in metalworking fluids.

The bacterium Mycobacterium immunogenum has been implicated in causing the lung condition hypersensitivity pneumonitis (HP) in factory workers exposed to colonized metalworking fluids (MWFs). M. immunogenum-specific, real-time quantitative PCR detection technique (MiRT-qPCR) was implemented on a large scale to 363 MWFs of varying types, originating from the United States and Europe, that had been in use for between 30 days and 1 year. In MWFs that contained between 10(3) and 10(9) culturable general heterotrophs mL(-1) the technique detected between 5 and 2 × 10(6) mL(-1) M. immunogenum cell equivalents (CE) in 12.2% (23 of 189) of U.S. samples and between 8 and 6 × 10(5) mL(-1) CE in 39.1% (68 of 174) of samples from Europe. In contrast, only three cultured presumptive mycobacterial isolates across all samples were confirmed as M. immunogenum. Implementation of the assay demonstrated its practicality and further emphasized the limitations of using cultivation alone. Interestingly, no M. immunogenum were detected in mineral oil-based Bio-Concept MWFs from the United States, while it was more commonly detected in used MWFs based on formaldehyde-releasing biocides than in MWFs free of formaldehyde-depot biocides.

Detection of Mycobacterium immunogenum by real-time quantitative Taqman PCR.

A quantitative real-time 5'-nuclease (Taqman) PCR technique was developed to specifically detect Mycobacterium immunogenum. rpoB-specific primers and Taqman probe were evaluated for detection of M. immunogenum DNA extracted from pure cultures and from industrial metal working fluids (MWFs). Specificity was confirmed and the sensitivity of detection of M. immunogenum genomic DNA was shown to be approximately 9 fg (2 cell equivalents). When tested on industrial metal working fluids from the UK and USA from which no M. immunogenum CFU were recovered, the assay detected between 3.4x10(1) and 1.9x10(4) cell equivalents (CE) per ml, and increased the detection rate over culture to 37.5% (12 of 32 samples). This assay provides a specific, sensitive and rapid method for the detection of M. immunogenum and is applicable within industry for the early detection of this human pathogen and to the possible prevention of hypersensitivity pneumonitis (HP) in workers.

Bacterial contamination of preserved and non-preserved metal working fluids.

Metal working fluids (MWFs) are important facilities for certain industrial processes. Besides their advantage concerning the effective technical applicability it is disadvantageous that they are excellent culture media for bacterial and fungal growth and may therefore act as an infectious source for employees getting in contact with them. As a matter of prevention most of MWFs are nowadays treated with preservatives to eliminate the contamination of possible human pathogenic bacteria and fungi. Own investigations were concerned with the objectives of the actual and long-term study of the bacterial contamination of preserved and non-preserved MWFs from different MWF systems, the development of a standardized bacterial biotope in non-preserved MWFs and its efficacy to eliminate bacteria which caused infectious diseases in humans, the development of bacterial resistance against preservatives in preserved MWFs and the occurrence of infections and allergies in employees working with MWFs. Our results show: high numbers of different bacteria can be found in non-preserved as well as preserved MWFs; bacteria with a possible human pathogenic potential (category 2, TRBA 466) could almost exclusively be found in preserved MWFs. Those bacteria besides others were able to develop resistance against biocides and could therefore be detected in preserved MWFs for weeks and months; non-preserved MWFs develop stable bacterial biotopes which can be controlled by an artificial inoculation of bacteria. Those biotopes are able to eliminate bacteria which induce infectious diseases in humans; there are only very few data published concerning MWF caused infections or allergies. According to our results of a questionnaire 10% of employees getting in contact with MWFs, complain skin irritations/reactions. There was no difference working with preserved or non-preserved MWFs nor a clear cut correlation to the MWF contact. These data may motivate for more intensive epidemiological studies in this field. The results of our investigations point out that taking into consideration safe working conditions non-preserved MWFs even contaminated with a standardized combination of waterborne bacteria like Pseudomonas spec. up to 10(8) CFU/ml are an alternative to preserved MFWs.

The MHC-haplotype influences primary, but not memory, immune responses to an immunodominant peptide containing T- and B-cell epitopes of the caprine arthritis encephalitis virus Gag protein.

In this report, we describe a short peptide, containing a T helper- and a B-cell epitope, located in the Gag protein of the caprine arthritis encephalitis virus (CAEV). This T-cell epitope is capable of inducing a robust T-cell proliferative response in vaccinated goats with different genetic backgrounds and to provide help for a strong antibody response to the B-cell epitope, indicating that it may function as a universal antigen-carrier for goat vaccines. The primary immune response of goats homozygous for MHC class I and II genes showed an MHC-dependent partitioning in rapid-high and slow-low responses, whereas the memory immune response was strong in both groups, demonstrating that a vaccine based on this immunodominant T helper epitope is capable to overcome genetic differences.