Secretome of the carcinogenic helminth Spirocerca lupi reveals specific parasite proteins associated with its different life stages.

Spirocerca lupi is a parasitic and carcinogenic nematode of canids distributed in tropical and subtropical regions around the world. The excretion-secretion proteins (PES) of S. lupi have been suggested to play a role in the pathogenesis of its infection. We aimed to identify the PES of different stages of S. lupi and search for proteins that would be useful for diagnostic, therapeutic and vaccination purposes as well as understand their functions. A nano-UPLC mass spectrometry de novo analysis was performed on proteins collected from cultures of S. lupi L3 larvae, L4 females, adult females and adult males from naturally infected hosts. A total of 211 proteins were identified in all cultures. Accordingly, 117, 130, 99 and 116 proteins were detected in L3 larva, L4 females, adult females and adult males, respectively, with a strong correlation in the biological replicates (Pearson coefficients > 0.73). Fourty-four proteins were detected in all developmental stages, 64 were stage-specific and 49 were exclusively identified in L4 females. Cell compartment enrichment analysis revealed that proteins common to all stages were cytoplasmatic (p < 9.x10-6), whereas L4 unique proteins were in collagen trimers, and macromolecular complexes (p < 0.00001). Functional enrichment analysis of proteins showed significant enrichment in lipid metabolism in L3-unique proteins (p<0.00005), in mannose metabolism and protein de-glycosylation for L4-unique proteins (p < 0.00004), and in phosphorus metabolism in proteins shared by all stages (p <  2.1 x10-9). Interestingly, annexin 6, associated with cancer in humans, was detected in all life stages, but in a larger abundance in L4 females and adults. These findings indicate that S. lupi establishes complex interactions with its hosts by an arsenal of proteins expressed in different patterns in each life stage which influence the pathogenesis and oncogenesis of S. lupi and may be used as potential targets for diagnostic assays, drug targets or vaccine candidates.

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

Equine thelaziosis caused by the eyeworm Thelazia lacrymalis is a parasitic disease transmitted by muscid flies. Although equine thelaziosis is known to have worldwide distribution, information on the epidemiology and presence of the intermediate hosts of T. lacrymalis is lacking. In the present work, a PCR-RFLP based assay on the first and/or second internal transcribed spacer (ITS1 and ITS2) of ribosomal DNA was developed for the detection of T. lacrymalis DNA in its putative vector(s). The sensitivity of the technique was also assessed. The restriction patterns obtained readily differentiated T. lacrymalis from four species of Musca (Diptera, Muscidae) (i.e. Musca autumnalis, Musca domestica, Musca larvipara and Musca osiris), which are potential vectors of equine eyeworms. The molecular assay presented herein is a useful tool to identify the intermediate host(s) of T. lacrymalis in natural conditions and to study its/their ecology and epidemiology.

Differentiation among three species of bovine Thelazia (Nematoda: Thelaziidae) by polymerase chain reaction-restriction fragment length polymorphism of the first internal transcribed spacer ITS-1 (rDNA).

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.