Mycoplasma bovis Serology as an Example
9. Conclusions
9.1 What has been achieved
Over the past 10 years the tremendous progress that has been made in the field of molecular biology have provided microbiologists with an increasing panel of techniques that have allowed the design of more refined and more effective diagnostic assays.
Recombinant proteins were soon adopted as key antigens in serological assays but their potential in routine diagnosis was compromised by the high background interference with host cell proteins. This was later overcome by the introduction of recombinant fusion proteins which facilitated the purification of the antigen of interest from the host cell proteins. Because viruses possess a rather simple and small genome, recombinant proteins have been widely used by virologists for instance to discriminate between closely related viruses, e.g., between HTLV-I and HTLV-II (Chen et al., 1990; Miyagawa et al., 1998) or to detect specific infections via the identification of circulating antibodies, e.g., to HIV (commercial recombinant HIV antigens are manufactured by several companies).
Concerning food-borne pathogens, several recombinant proteins have been produced for the improvement of diagnostic assays either to detect circulating host antibodies to a given pathogen or to detect antigens by immuno-capture assays. Some examples include the production of truncated forms of Listeriolysin O as a specific and sensitive antigen for serodiagnosis of listeriosis (Gholizadeh et al., 1996), the production of recombinant antigens encoded by the LEE locus of E. coli O157 for the detection of patients infected with verocytotoxin-producing E. coli (Jenkins et al., 2000), or the production of recombinant fimbrial proteins for the specific detection of Salmonella enteritidis infection in poultry (Rajashekara et al., 1998, 1999). Within a period of six months, two independent reports have described the potential of different recombinant antigens for the detection of Toxoplasma gondii infection (Lecordier et al. 2000; Li et al. 2000).
9.2 What has been neglected
The production of recombinant antigens in clinical microbiology has rapidly increased over the past few years and their use as diagnostic or epidemiological tools in ELISA is more and more accepted as long as validation tests are progressing along to ascertain their performance in the field. However, the production of specific MAbs or monospecific polyclonal sera directed towards an amino acid sequence specific of a given pathogen or serovar using recombinant fusion antigens has been neglected. This is rather surprising, specially when sequence data are already available. Advantages in using recombinant proteins for this purpose include the facts that they (i) display a limited repertoire of epitopes compared to more complex preparations, (ii) can be easily purified avoiding for instance difficulties in generating enough material from columns or gels for immunization, and (iii) facilitate the screening of the appropriate MAb. For instance, using a recombinant construct of the VlpA product of the swine pathogen Mycoplasma hyorhinis, a specific rabbit antiserum has been produced that can distinguish this particular product from the seven other members of the Vlp family with which VlpA shares several antigenic motifs (Citti and Wise, 1995). Overall, this strategy could certainly benefit to the development of specific reagents for bacterial typing.
Christine Citti, Marion Brank and Renate Rosengarten
9.3 What needs to be done
The ultimate goal of a given diagnostic assay is to detect a particular infection before apparition of the clinical symptoms and before it spreads to other healthy animals.
Consequently it has to be specific and sensitive. As mentioned above, new reagents for the improvement of these assays are currently emerging or are already available for a large number of pathogens. The critical question which in most cases remains to be answered is how do they compare and compete with existing serological tests already in use in diagnostic laboratories. Validation in the field of these new assays with reference material followed by standardization among laboratories as well as the definition of which type of assay will answer accurately and reproducibly a particular diagnostic question are immediate and future challenges.
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