Molecular characterization of Thelazia lacrymalis (Nematoda, Spirurida) affecting equids: a tool for vector identification

The nucleotide sequence of the second internal transcribed spacer (ITS-2) from ribosomal DNA has been determined for 3 members of the Hypodontus macropi species complex. Sequences were compared from nematodes collected from 3 species of Australian macropodid marsupial, Petrogale persephone, Macropus robustus robustus and Thylogale billardierii. The ITS-2 of each operational taxonomic unit ranged from 287 to 292 bases in length, and had a GC content of 36.6-40.1%. Differences in nucleotide sequence between nematodes from the different host species ranged from 25.0% to 28.3%. The data suggest that H. macropi from P. persephone represents a different species to those in M. r. robustus and T. billardierii. The unique feature of this study is that it represents a comparison of the ribosomal DNA sequences of nematode species which are morphologically indistinguishable but which have been demonstrated to be genetically distinct (i.e. cryptic) species based on electrophoretic data. The results also demonstrate further that morphological characters alone are often not adequate for species recognition. Differences between these 3 species of H. macropi in their recognition sites for restriction endonucleases, indicates that a PCR-RFLP approach could be used, in conjunction with allozyme electrophoresis, to establish how many species are present within the H. macropi complex.

The internal transcribed spacer region (ITS1, 5.8S gene and ITS2) of the two filarial nematodes Onchocerca volvulus and Mansonella ozzardi was sequenced, and two species-specific primers designed in the ITS2 to develop a PCR-based method for their specific detection and differentiation. When used with a universal reverse primer, the two species-specific primers gave amplification products of different size, which were readily separated in an agarose gel. The PCR was tested on skin biopsies from 51 people from three localities in Brazil where M. ozzardi is present, and results have been compared with those of parasitological examination of blood. The species-specific PCR gave a higher percentage of detection of infection by M. ozzardi than the parasitological examination of blood. No infection with O. volvulus was detected by PCR. This PCR-based assay may assist in determining the nature of infection in areas where both filarial species exist in sympatry.

Thelazia gulosa, Thelazia rhodesi and Thelazia skrjabini are nematodes transmitted by some species of Musca (Diptera: Muscidae) which cause ocular infestations in bovines. Differences in the rDNA of these species were determined by a PCR using different sets of relatively conserved oligonucleotide primers. PCR on the first internal transcribed spacer (ITS-1) revealed differences in size in Thelazia species (437 bp for T. gulosa, 370 bp for T. rhodesi and 506 bp for T. skrjabini) while the DNA control of Musca spp. was not amplified. The ITS-1 amplicons of the three species were sequenced and then analysed. The GC contents ranged from 26 to 36% and the level of differences in the nucleotide sequences of ITS-1 was lower between T. skrjabini and T. gulosa (39%) than the latter and T. rhodesi (49-56%). Restriction fragment length polymorphism (RFLP) of ITS-1 amplicons was also carried out and the restriction profiles compared. Clear genetic differences among the three Thelazia examined were demonstrated by using the enzymes HpaII, CpoI and SspI. This PCR-RFLP for the delineation of T. gulosa, T. rhodesi and T. skrjabini offers prospects as a molecular epidemiological tool to study parasite transmission patterns and prevalence.