First genetic characterisation and phylogenetic analysis of Trichomonadida from Gallus gallus domesticus (Aves: Phasianidae) and its nematode parasite Heterakis gallinarum (Ascaridida: Heterakidae) in Tunisia

Flagellated protozoa of the order Trichomonadida infect a variety of vertebrates, including poultry such as Gallus gallus domesticus (Linnaeus). Several trichomonad pathogens are of significant veterinary importance due to their role in diseases that cause high mortality rates in chickens. Despite the importance of Trichomonadida in poultry health, molecular studies on these protozoa in North Africa are limited. This study addresses this gap by investigating the genetic diversity and evolutionary relationships of Trichomonadida isolated from G. gallus domesticus and its nematode parasite Heterakis gallinarum (Schrank, 1788) in Tunisia, using a multilocus molecular approach with 18S rRNA and α-actinin 1 genes. Based on both markers, all Tunisian haplotypes, which clustered with those from France, were found to belong to genotype2. 18S rRNA analysis revealed the existence of protozoans such as Histomonas meleagridis (Smith, 1895) and Parahistomonas wenrichi Lund, 1963 in coinfection with H. gallinarum, confirming a possible mixed infection. Additionally, when analysing caecal samples, other Trichomonadida species were identified, including Simplicimonas sp. and Tetratrichomonas gallinarum (Martin et Robertson, 1911). These findings suggest a complex protozoan community within the studied hosts. Phylogenetic analysis revealed a close relationship between H. meleagridis and P. wenrichi, as well as between Simplicimonas sp. and the Monoceromonas-Tritrichomonas group. Both H. meleagridis genotypes 1 and 2 exhibited a sister-group relationship with P. wenrichi, with strong support for a common evolutionary origin. Tetratrichomonas gallinarum was basal in the tree, suggesting early divergence in the Trichomonadida lineage. This study provides, for the first time, insights into the genetic diversity of trichomonadids in Tunisia. The 18S rDNA locus proved to be effective for assessing the genetic diversity of H. meleagridis, P. wenrichi, T. gallinarum and Simplicimonas sp. and showed a possible mixed infection. The findings provide valuable insights into the genetic characteristics of these parasites in Tunisian poultry farms and contribute to the understanding of Trichomonadida diversity, enhancing disease control and prevention efforts.