Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.

Exposure and risk assessment in occupational exposure to fungi: aspects to consider in highly contaminated settings

Although a clear correlation between levels of fungi in the air and health impacts has not been shown in epidemiological studies, fungi must be regarded as potential occupational health hazards. Fungi can have an impact on human health in four different ways: (1) they can infect humans, (2) they may act as allergens, (3) they can be toxigenic, or (4) they may cause inflammatory reactions. Fungi of concern in occupational hygiene are mostly non-pathogenic or facultative pathogenic (opportunistic) species, but are relevant as allergens and mycotoxins producers. It is known that the exclusive use of conventional methods for fungal quantification (fungal culture) may underestimate the results due to different reasons. The incubation temperature chosen will not be the most suitable for every fungal species, resulting in the inhibition of some species and the favouring of others. Differences in fungi growth rates may also result in data underestimation, since the fungal species with higher growth rates may inhibit others species’ growth. Finally, underestimated data can result from non-viable fungal particles that may have been collected or fungal species that do not grow in the culture media used, although these species may have clinical relevance in the context. Due to these constraints occupational exposure assessment, in setings with high fungal contamination levels, should follow these steps: Apply conventional methods to obtain fungal load information (air and surfaces) regarding the most critical scenario previously selected; Guideline comparation aplying or legal requirements or suggested limits by scientific and/or technical organizations. We should also compare our results with others from the same setting (if there is any); Select the most suitable indicators for each setting and apply conventional-culture methods and also molecular tools. These methodology will ensure a more real characterization of fungal burden in each setting and, consequently, permits to identify further measures regarding assessment of fungal metabolites, and also a more adequate workers health surveillance. The methodology applied to characterize fungal burden in several occupational environments, focused in Aspergillus spp. prevalence, will be present and discussed.