2. Universidad Cardenal Herrera-CEU

Oropharyngeal avian trichomonosis is mainly caused by Trichomonas gallinae, a protozoan parasite that affects the upper digestive tract of birds. Lesions of the disease are characterized by severe inflammation which may result in fatality by starvation. Two genotypes of T. gallinae were found to be widely distributed in different bird species all over the world. Differences in the host distribution and association with lesions of both genotypes have been reported. However, so far no distinct virulence factors of this parasite have been described and studies might suffer from possible co-infections of different genotypes. Therefore, in this paper, we analyzed the virulence capacity of seven clones of the parasite, established by micromanipulation, representing the two most frequent genotypes. Clones of both genotypes caused the maximum score of virulence at day 3 post-inoculation in LMH cells, although significant higher cytopathogenic score was found in ITS-OBT-Tg-1 genotype clones at days 1 and 2, as compared to clones with ITS-OBT-Tg-2. By using one representative clone of each genotype, a comparative proteomic analysis of the membrane proteins enriched fraction has been carried out by a label free approach (Data available via ProteomeXchange: PXD013115). The analysis resulted in 302 proteins of varying abundance. In the clone with the highest initial virulence, proteins related to cell adhesion, such as an immuno-dominant variable surface antigen, a GP63-like protein, an armadillo/beta-cateninlike repeat protein were found more abundant. Additionally, Ras superfamily proteins and calmodulins were more abundant, which might be related to an increased activity in the cytoskeleton re-organization. On the contrary, in the clone with the lowest initial virulence, larger numbers of the identified proteins were related to the carbohydrate metabolism. The results of the present work deliver substantial differences between both clones that could be related to feeding processes and morphological changes, similarly to the closely related pathogen Trichomonas vaginalis.

Extensive diversity and even new species have been described within the avian oropharyngeal trichomonad complex in recent years. In this study we developed clonal cultures from four isolates selected by their different ITS1/5.8S/ITS2 (ITS) genotype and their association with gross lesions of avian trichomonosis. Isolates were obtained from an adult racing pigeon (Columba livia) with clinical signs of avian trichomonosis, a juvenile wood pigeon (Columba palumbus) and an European turtle dove (Streptopelia turtur) without clinical signs, and a nestling of Eurasian eagle owl (Bubo bubo) with gross lesions. Multi-locus sequence typing analysis of the ITS, small subunit of ribosomal rRNA (SSUrRNA) and Fe-hydrogenase (Fe-hyd) genes together with a morphological study by optical and scanning electron microscopy was performed. No differences in the structures were observed with scanning electron microscopy. However, the genetic characterisation revealed three novel sequence types: one for the SSUrRNA region and two for the Fe-hyd gene. Clones from the Eurasian eagle owl and the wood pigeon were identified as Trichomonas gallinae analysing each locus, with an ITS genotype of T. gallinae-1 and T. gallinae-2 groups, respectively, which are commonly reported in wild birds from Europe and America. Clones of trichomonads from the racing pigeon and European turtle dove showed higher similarity with Trichomonas tenax and Tetratrichomonas canistomae than with T. gallinae strains at their ITS region, respectively. SSUrRNA sequences grouped clones in a T. gallinae, T. tenax and T. canistomae clade. Further diversity of T. gallinae was detected within the Fe-hyd locus. Morphometric comparison by optical microscopy with clonal cultures of T. gallinae (T. gallinae-1 and T. gallinae-2 ITS genotypes), revealed significant statistical differences on axostyle projection in the clone from European turtle dove, which was 1.6 μm longer (mean 8.2 μm) than the mean values reported for T. gallinae (6.4 μm). Possible new species within the Trichomonas genus were detected in isolates obtained from the racing pigeon and the European turtle dove, but further reports will be needed to confirm their host distribution.