Contaminations contaminate common databases.

The polymerase chain reaction (PCR) is a very powerful method to detect and identify pathogens. The high sensitivity of the method, however, comes with a cost; any of the millions of artificial DNA copies generated by PCR can serve as a template in a following experiment. If not identified as contaminations, these may result in erroneous conclusions on the occurrence of the pathogen, thereby inflating estimates of host range and geographic distribution. In the present paper, we evaluate whether several published records of avian haemosporidian parasites, in either unusual host species or geographical regions, might stem from PCR contaminations rather than novel biological findings. The detailed descriptions of these cases are shedding light upon the steps in the work process that might lead to PCR contaminations. By increasing the awareness of this problem, it will aid in developing procedures that keep these to a minimum. The examples in the present paper are from haemosporidians of birds, however the problem of contaminations and suggested actions should apply generally to all kinds of PCR-based identifications, not just of parasites and pathogens.

Identification of Fasciola hepatica recombinant 15-kDa fatty acid-binding protein T-cell epitopes that protect against experimental fascioliasis in rabbits and mice.

Vaccination with fatty acid-binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Thl/Th2 response to vaccination.

Toxocara canis antigens stimulate the production of nitric oxide and prostaglandin E2 by rat alveolar macrophages.

The effect of four Toxocara canis antigens on nitric oxide (NO) and prostaglandin E2 (PGE2) synthesis was studied in vitro using rat alveolar macrophages. Somatic and excretory/secretory T. canis antigens prepared from adult worms and LII larvae were incubated with rat alveolar macrophages obtained by bronchoalveolar lavage at concentrations of 0.1-50 microg/ml. Both excretory/secretory adult antigen (ESA) and somatic LII antigen (SLII) stimulate the release of nitrites by alveolar macrophages. This effect was specific (inhibited by L-NAME and L-canavanine) and dose-dependent; 30 microg and 10 microg being the most effective concentrations of ESA and SLII, respectively. Western blot and reverse transcriptase-polymerase chain reaction analyses revealed that ESA antigen stimulates the production of NO at transcriptional level. T. canis ESA also stimulated macrophages to produce PGE2 at transcriptional level. The addition of L-canavanine decreased the release of PGE2 significantly, which suggests that NO mediates the production of this prostaglandin. These results indicate that T. canis can stimulate the release of vasodilatory mediators by macrophages of the host.