Pyrosequencing analysis identifies discrete populations of Haemonchus contortus from small ruminants

The genus Haemonchus consists of blood-sucking parasitic nematodes in the abomasum of ruminants. Members of this genus are responsible for extensive production losses, particularly of small ruminants in the tropics but are also found in temperate regions. In this study, we examined the internal transcribed spacers-1 and -2 of rRNA in Haemonchus spp. The rRNA region spanning the internal transcribed spacers-1, -2 and the 5.8S rRNA gene was amplified by PCR from each of 10 worms from Swedish sheep, a Swedish goat and Kenyan sheep. The fragments were sequenced and examined with respect to genetic differences fixed among the three isolates. These and additional worms were further analysed with Pyrosequencing technology. This technique allowed us to rapidly analyse 110 individuals in three putative polymorphic nucleotide positions that were initially identified with dideoxy sequencing. The geographical isolates could to a large extent be genetically distinguished, but none of the polymorphic positions were consistent among all individuals within each isolate. Furthermore an alignment of our sequences and a consensus sequence published for Haemonchus contortus revealed two differences in positions 123 and 196 in internal transcribed spacers-2. Although these positions were previously reported as heterogenic, no polymorphism was detected among the 30 worms sequenced in the present study. Modelling of the internal transcribed spacers-2 secondary structure based on our data also identified a new putative long-range interaction. The isolates are best described as populations. In conclusion, consistent differences were not identified and the studied isolates are therefore best described as discrete populations. This study also reveals for the first time the potential of Pyrosequencing technology as a tool in the analysis of nematode population genetics.