PRESENTACIÓN DE OFERTAS (Art 53 RLCE) a) En ACTO PÚBLICO.

Sibley and Boothroyd (1992a) using PCR-RFLP with a probe derived from a repetitive genomic fragment (BS), suggested a clonal lineage for 10 virulent strains o f T. gondii isolated from humans and animals, all of which are considered mouse- virulent (Sibley and Boothroyd, 1992a). These 10 Z gondii virulent strains had an identical genotype at three genomic loci investigated {SAGI, 850, BS). They demonstrated a correlation between RFLP patterns and strain virulence for mice. But 18 avirulent strains had moderately polymorphic RFLP patterns different from those of the virulent strains.

Conversely, the RFLP patterns generated by using different DNA probes in other studies (Cristina et al, 1991, Parmley et al; 1994b) did not correlate with virulence. Using two repetitive probes (TGRIE and TGR6), Cristina et al, (1995) revealed that three virulent strains are closely related, giving similar RFLP patterns. However, the virulent strain (MAS) had completely different RFLP patterns from those o f the other three virulent strains. It seems that virulent strains, like avirulent strains may be polymorphic when different strains and larger number o f different loci were studied.

RFLP has also been used to analyze the different laboratory stocks of the RH strain revealing genetic heterogeneity amongst them (Howe & Sibley, 1994). Parmely et al (1994b) reported a limited usefulness of anti-p22 Mb to identifying virulent and avirulent T. gondii strains and suggested that PCR may be more useful for detecting and typing T gondii strains than immunological techniques.

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If RFLPs are chosen in regions of protein coding sequence, then polymorphisms as detected by PCR are more likely to correlate with the phenotype of the strain. RPLPs in non-coding regions where most nucleotide changes are tolerated without affecting the phenotype may be less useful. By studying divergence in protein coding sequences, it might be possible to develop a phylogenetic tree that allows related strains to be grouped and the differences within groups studied.

1.7. 4 rRNA Riboprinting

The main function of rRNA is protein synthesis, so it is not surprising that the genomes of all organisms contain sequences that code for these essential molecules. Analyses of rRNA genes have been facilitated by the fact that portions o f the coding region evolve very slowly. Others evolve some what more rapidly and provide phylogenetic markers at intermediate evolutionary conserved loci across widely different organisms which has opened the possibility for development o f what Wheelis et al, (1992) referred to as a ‘global classification’ for all of life.

RNA genes in the nuclei o f eukaryotic cells usually exist as tandemly repeated elements, each repeat unit composed o f a highly conserved coding sequence of total length about 6 Kb, plus shorter and more variable noncoding spacer regions. The repeated rDNA molecules may occur at one or more chromosomal sites. The copy number o f rDNA repeats per genome varies from several hundred in some mammals and insect to many thousands in plants (Long and Dawid, 1980).

According to Guay et al, (1992), T. gondii has about 110 copies of 16S-like rDNA per genome that can be used for studying intra individual polymorphism. Coding regions located for the 16S-like rDNAs have gained widespread acceptance for molecular systematics (Gagnon et al, 1993). Analysis of 16S-like rRNAs is a promising approach to observe the polymorphisms between T. gondii isolates and closely related species. The result of studies on 16S-like rDNA will be discussed in chapter 6 of this thesis.

1.7. 5 Random Amplified Polymorphism DNA (RAPD)

This technique was first described independently at the same time by Williams et al, (1990) and Welsh and McCleland (1990), and termed random amplified polymorphism DNA (RAPD) analysis. It is based on random amplification of DNA fragments by the use o f a single short primer (commonly 10 mers) with an arbitrary sequence. The single primer will support DNA amplification from the genomic template if binding sites on opposite strands of the template exist within a distance that can be reversed by DNA polymerase, up to several thousand nucleotides (Innis et al, 1990). Separation o f the PCR products on agarose gels and visualization of the DNA bands with ethidium bromide staining can generate fingerprint patterns and specific fragments may be present as unique bands. This method detects abundant polymorphism, which can be used for genetic mapping application, genetic diagnosis and for genetic comparison of a large range o f organisms (Williams et al, 1993). The nature of amplified fragments is highly dependent on the primer sequence and on the DNA sequence of the genome being

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assayed. Genomic polymorphisms at one or both primer binding sites may result in disappearance o f the amplified bands.

According to Guo and Johanson (1995 a, b), Mackensted and Johanson (1995) and Stevens and Tibayemc (1995), RAPD-PCR is an appropriate technique in the detection o f polymorphisms randomly distributed in the genome among very closely related organisms, including parasitic protozoa without the need for predetermined sequence information. RAPD- PCR also has the additional advantages of technical simplicity, speed, high resolution and the requirement for only small amounts of DNA (about 20 ng DNA per reaction). However despite the advantages mentioned above there are some concerns about reproducibility of the results obtained by this method, particularly with contamination by host DNA.

Guo and Johnson (1995a) observed significant genetic heterogeneity among 11 r. gondii strains with different virulence for mice. The six murine virulent T. gondii strains formed one group and the five avirulent formed another. These results suggested that T. gondii might actually comprise o f two major clonal lineages, correlated with virulence.

Further studies performed by Guo and Johnson (1996) utilizing 18 primers and 35 r. gondii strains, confirmed the previous result. The two T. gondii clonal lineages found are consistent with the fact that strains with similar virulence types form two defined subsets which have probably evolved independently following their initial separation. The two virulent and avirulent populations in both lineages appear to consist o f a range of strains with a similar level of genetic diversity. In contrast, Boothroyd and Sibley (1992a) found that virulent strains comprise o f a

single clonal lineage while avirulent strains are moderately polymorphic, consistent with the finding o f Cristina et al, (1995) in which the TGRIE was used as a PCR target. Guo and Johnson (1996) identified primers, which may differentiate between mouse virulent and avirulent T. gondii strains. In more recent study Guo and Johnson (1997) described primer B 12 as the producer of virulence-specific fragment and primers B5, C8, and C20 as producer avirulence-specific fragments. Once again these result confirmed the previous result that genus Toxoplasma may contain two clonal lineages directly correlated with mouse virulence.

1.8 Summary

Strain variation is an important issue in T. gondii biology and pathogenesis. There is evidence that suggests virulence and development o f encephalitis during chronic infection varies among T. gondii strains (Suzuki et al, 1989). In addition, it has been shown that chronically infected mice died as a result of active toxoplasmosis when re-infected by another strain o f T. gondii Thus, immunity to T. gondii infection may be strain-specific (Araujo et al, 1997). It has been suggested that toxoplasmic encephalitis could be more common in AIDS patients who are infected with persistent cysts o f group B (Type II) o f T gondii strains (Gross et al, 1997b). This group can produce more cysts in infected individuals due to increase potential to convert into bradyzoites. Therefore, it is becoming increasingly important to identify the nature o f the T. gondii strains in clinical infections, facilitating early characterization prior to culture selection. These aims have been set for more investigation in strain variation between T. gondii isolates.

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To establish the phenotypic differences:

1 Compare the growth characteristics o f T. gondii strains in in vitro cell cultures such as, Human Embryonic Fibroblast (HEL) and THPl monocytes. 2 Adapt an in vitro model to study strain variation in T. gondii isolates.

3 Improve the long-term preservation system by optimizing ciyo-preservation. 4 Morphological observation o f the tachyzoite stage by transmission

electronmicroscopy and scanning electron microscopy.

To assess the selected gene as a potential genetic marker for studying stain variation directly on clinical samples.

5 Study the SAGA gene as a marker o f strain variation 6 Study the B \ gene as a marker for strain variation

7 Study rDNA using PCR-RFLP, PCR-SSCP and sequencing to search for polymorphism.

8 Application o f findings to other T. gondii isolates and clinical specimens in order to examine the suitability for strain typing

The aim o f this thesis is to establish rapid methods for classification o f the pathogenic potential of clinical isolates of T. gondii.