Tunisian Toxoplasma gondii strains genotyping by the use of AK69 marker

In our study, the contribution of sequence polymorphisms in the AK69 gene as typing markers for T. gondii was investigated for the first time in an epidemiological study. The coding region of the marker was amplified, sequenced and aligned for different Toxoplasma strains. The identified nucleotide polymorphism at 12 positions was able to highly discriminate between the different congenital toxoplasmosis Tunisian strains. Moreover the high detection sensitivity level of the marker enabled unambiguous identification of mixed/recombinant genotypes directly.It can be, thus, very useful for direct typing in areas where such genotypes are frequently encountered, mainly in the African continent.Toxoplasma gondii (T. gondii) is an intracellular protozoan that infects all warm-blooded animals including humans. It is considered as one of the most widespread parasites in the world [1]. While three genetic lineages I, II and III are predominant in Europe and North America [2], recombinant strains, are rather more frequently observed in North Africa [3].Different methods are used for genotypic characterization of T.gondii [4,5]. A collection of more than 200 markers, allowing direct genotyping, was described [6]. Nevertheless most of them are biallelic and could distinguish just two of the 3 clonal types at a single locus. Only a small percentage of markers presents two biallelic polymorphisms that lie closely by each other and are able to identify even recombinant strains. AK69, one of these markers, located on chromosome X, is the only one supposed to distinguish between the different types using a single restriction enzyme [6].The aim of this study is to investigate the AK69 marker performance and contribution to the direct characterization of recombinant strains in Tunisia.This study included 3 reference strains: RH (Type I), PRU (Type II) and NED (Type III) provided by CRB Toxoplasma (France) and 14 clinical specimens (11 Amniotic Fluids, 2 Placentas and 1 new born Cerebra