EVIDENCE OF P-GLYCOPROTEIN SEQUENCE DIVERSITY IN CYATHOSTOMINS

Abstract

P-glycoproteins (Pgps) are adenosine triphosphate-binding transporter proteins thought to be associated with multi-drug resistance in mammals and protozoans and have been suggested to be involved in the mechanism of ivermectin (IVM) resistance in Haemonchus contortus. Until now, resistance to IVM has not been reported in cyathostomins in horses in spite of its widespread and frequent use. Reasons for this might be differences in the molecular mechanism of the development of resistance. Based on this hypothesis, the present study was carried out to find homologues of Pgp in cyathostomins. A 416-bp polymerase chain reaction (PCR) product was generated using complementary DNA (cDNA) of Cylicocyclus elongatus and Cylicocyclus insigne and degenerate primers, located in the conserved Pgp nucleotide-binding domains. Resulting PCR products showed interspecific nucleotide and amino acid sequence identities of 73.3 and 76.8%, respectively. Specific primers were designed based on the Cc. elongatus sequence, and a PCR product of 268-bp was amplified from cDNA of single adults of Cylicocyclus radiatus, Cc. insigne, Cylicocyclus nassatus, Cc. elongatus, Cylicostephanus hybridus (2 individuals), Cylicostephanus goldi, Cyathostomum pateratum, Cyathostomum coronatum, and Cyathostomum catinatum. Two clusters of sequences were found representing 2 different internucleotide-binding domains (IBDs). A further distinct IBD is represented by the 416-bp PCR product of Cc. insigne. Therefore, a total of 3 clearly different sequences of the IBD were cloned and sequenced, suggesting that at least 2 Pgp genes exist in cyathostomins.