Faculty of Veterinary Medicine University of Zagreb
Abstract
Microsatellite DNA polymorphisms can be utilised to assess intra-specific genetic diversity and hence are useful for characterisation of species and strains of trypanosomes. Here, we present four new microsatellite markers specific for T. vivax isolated from Ugandan cattle and goats. The GeneDB partial shotgun 5x coverage sequence of T. vivax available as of 1st August 2005 was used and targeted the genomic sequence of T. vivax that has no cross amplification with other livestock-infective trypanosomes. Only di-; tri-; tetra;- and pentanucleotide microsatellites not less than five units were selected. Although pentanucleotide repeats on screening appeared to have the desired variability, they gave poorer PCR products compared to di-, tri- and tetranucleotide repeats. Mononucleotide repeats presented difficulty in detecting visible bands on agarose gels from their amplification and were omitted from this study. Clear length polymorphism was obtained with guanine, thymine and adenine repeated 16 times (GTA)16 while cytosine, adenine, cytosine and thymine (CACT)15 gave size and length variability. Bands of similar size were obtained from thymine and adenine (TTA)24 microsatellite, approximately 150 base pairs long and 180-200 base pairs from the cytosine and adenine (CA)26 microsatellite. These findings suggest that different subtypes of T. vivax exist in Uganda; the polymorphic forms derived from microsatellite band size differences may suggest this parasite exhibits virulence differences as has been shown in T. Congolense subtypes.Polimorfizam mikrosatelitske DNA može se rabiti za procjenu unutarvrsne genetske raznolikosti pa tako i za karakterizaciju vrsta i sojeva tripanosoma. Prikazana su četiri nova mikrosatelitska markera specifična za vrstu T. vivax izdvojenu iz goveda i koza u Ugandi. GenDB kratka i specifična sekvencija T. vivax dostupna nakon 1. kolovoza 2005. bila je ciljano rabljena za određivanje genomskoga slijeda za protozoon T. vivax koji nije pokazivao križne reakcije s drugim tripanosomama zaraznima za stoku. Izabrani su bili samo di-, tri-, tetrai pentanukleotidni mikrosateliti. Premda se činilo da pentanukleotidne ponavljajuće sekvencije u probirnom testu imaju potrebnu varijabilnost, one su dale lošije PCR proizvode u odnosu na di-, tri- i tetranukleotidne ponavljajuće sekvencije. Mononukleotidne ponavljajuće sekvencije nisu dale jasno vidljive trake na agaroznom gelu pa nisu bile dalje istražene. Jasan polimorfizam postignut je upotrebom gvanina, timina i adenina sa šesnaesterostrukim ponavljanjem (GTA)16 dok je sekvencija citozin, adenin, citozin i timin (CACT)15 bila varijabilna u odnosu na veličinu i dužinu. Sekvencije slične veličine bile su dobivene od mikrosatelita koji su sadržavali timin i adenin (TTA)24, a one od 150 parova baza te 180 - 200 parova baza od mikrosatelita citozina i adenina (CA)26. Ovi nalazi govore u prilog postojanju različitih podtipova protozoona T. vivax u Ugandi, koji bi se mogli odlikovati i različitom virulencijom kao što je dokazano za podtipove T. congolense
