Understanding the role of Microbial Parasites and Microbiome Interactions in Iraq, Türkiye, and Algeria under One Health perspective

Intestinal microbial eukaryotic parasites constitute a substantial public health and veterinary burden worldwide, particularly in low- and middle-income countries, where close contact between humans, livestock, and the environment facilitates complex transmission pathways. Despite their recognised zoonotic and environmental significance, the epidemiology, genetic diversity, and transmission dynamics of key intestinal protozoa and microsporidia remain insufficiently characterised in many regions. This doctoral research adopts an integrated One Health framework to investigate the occurrence, molecular diversity, host associations, and ecological interactions of Cryptosporidium spp., Giardia duodenalis, Blastocystis sp., Enterocytozoon bieneusi, and Entamoeba histolytica across human, animal, and environmental compartments in selected rural settings in Iraq, Türkiye, and Algeria. The work comprises a series of cross-sectional and prospective molecular epidemiological studies conducted in geographically and socio-ecologically distinct rural communities. Samples were collected from humans, livestock (including cattle, sheep, goats, and equines), and environmental matrices (water, soil, mud, and lake water). Across studies, faecal samples from humans (n ranging from 25 to 124), livestock (n ranging from 50 to over 300), and equines (n = 197), alongside environmental samples (n up to 40), were analysed using a combination of light microscopy, culture, quantitative polymerase chain reaction (qPCR), nested PCR, and Sanger sequencing. Molecular characterisation targeted established genetic loci, including the small subunit ribosomal RNA (SSU rRNA), gp60, beta-giardin (bg), triosephosphate isomerase (tpi), and internal transcribed spacer (ITS) regions, enabling species identification, genotyping, and subtyping. Selected studies further incorporated high-throughput 16S rRNA gene amplicon sequencing to explore associations between parasitic colonisation and gut microbiome composition. Across all study settings, intestinal protozoa were highly prevalent and genetically diverse. In rural Iraq, Blastocystis sp. and Cryptosporidium spp. were the most frequently detected microeukaryotes across humans, livestock, soil, and water, with evidence of overlapping host and environmental presence suggesting active zoonotic and environmental transmission. Molecular analyses identified Cryptosporidium parvum in both humans and sheep, highlighting zoonotic risk, while Cryptosporidium ubiquitum and Enterocytozoon bieneusi genotypes BEB6 and COS-I were detected exclusively in sheep, supporting their role as potential reservoirs. Several Blastocystis subtypes were identified, including ST1 in humans and ST4 and ST10 in sheep. Notably, microscopy consistently yielded higher detection rates for Blastocystis than molecular methods, underscoring methodological challenges in accurately assessing its prevalence. Entamoeba histolytica was not detected in any Iraqi samples. In Türkiye, large-scale One Health investigations revealed a high prevalence and extensive subtype diversity of Blastocystis sp. in humans, livestock, and environmental samples. Subtypes ST1-ST4 predominated in humans, with ST3 most frequent, while livestock were mainly colonised by ST10, alongside a remarkable diversity of additional subtypes, including ST24, ST25, and ST26, some reported for the first time in the country. Statistical analyses demonstrated a significant association between Blastocystis colonisation and host body mass index, with lean individuals exhibiting higher carriage rates. Integration of parasitological and microbiome data revealed subtype-specific interactions with gut bacterial communities; contrary to previous assumptions, ST4 colonisation was associated with reduced bacterial diversity and distinct compositional shifts, highlighting the ecological complexity of host-parasite-microbiota interactions. Parallel One Health studies in Türkiye focusing on Cryptosporidium spp. and Giardia duodenalis demonstrated widespread distribution across humans, livestock, and environmental samples. Giardia duodenalis assemblages A, B, and E were identified, including the first documented detection of assemblage E in a human host in Türkiye, challenging traditional assumptions regarding host specificity. Cryptosporidium species diversity was considerable, encompassing C. parvum, C. andersoni, C. ryanae, C. ubiquitum, and C. bovis, with C. parvum emerging as the dominant zoonotic species across hosts and environmental matrices. Household-level analyses revealed that the majority of households were positive for at least one parasite, emphasising intense local transmission pressure. While overall gut microbiome diversity did not differ significantly between infected and uninfected individuals, parasite-specific alterations in bacterial composition were observed, suggesting subtle but biologically relevant microbial interactions. Investigations into Enterocytozoon bieneusi under a One Health framework in Türkiye and Algeria revealed contrasting epidemiological patterns. In rural Türkiye, E. bieneusi was detected exclusively in livestock, with no human or environmental positives, yet zoonotic genotypes such as BEB6, J, and Type IV were identified alongside novel genotypes, indicating latent zoonotic potential. Phylogenetic analyses placed detected genotypes within Groups 1 and 2, both associated with human infection elsewhere. In Algeria, extensive surveys in cattle revealed a substantial prevalence of E. bieneusi, with multiple zoonotic genotypes clustering predominantly within Group 1, reinforcing the role of cattle as significant reservoirs of human-infective strains. Host age, sex, and farm composition significantly influenced infection risk, while production system and diarrhoeal status did not. Equine-focused studies in Algeria addressed critical gaps in knowledge regarding cryptosporidiosis, giardiasis, and blastocystosis in North Africa. Molecular screening of horses, donkeys, ponies, mules, and zebras revealed a high prevalence of Cryptosporidium spp., moderate prevalence of Blastocystis sp., and lower prevalence of Giardia duodenalis. Genotyping identified zoonotic C. parvum, equine-adapted C. equi, C. muris, and mixed C. parvum/C. hominis infections, including the first reports of C. equi subtypes in Algerian horses and C. hominis in donkeys. Giardia duodenalis assemblages could not be determined due to sequencing failure, representing a key limitation. Blastocystis subtyping revealed extensive diversity, including ST1-ST4, ST25, ST33, ST48-like sequences, and ST10-like clades, with evidence of mixed infections and novel subtype variants. Although several host and environmental factors were associated with infection in univariate analyses, none remained significant after multivariate adjustment, reflecting the multifactorial nature of transmission. Across all studies, mixed infections were common in both humans and livestock, particularly involving combinations of Blastocystis sp., Cryptosporidium spp., and Giardia duodenalis, although triple infections were not observed. Importantly, many infections were asymptomatic, underscoring the hidden burden of intestinal protozoa and their potential for silent transmission. Prevalence estimates based on sequence-confirmed detections were conservative, as non-target PCR amplifications were excluded, further emphasising the robustness of the reported findings. Collectively, this doctoral research provides a comprehensive and integrative assessment of intestinal protozoan and microsporidian infections across human, animal, and environmental interfaces in the Middle East and North Africa. The findings highlight substantial parasite burden, pronounced genetic heterogeneity, frequent co-infections, and compelling evidence of zoonotic and environmental transmission pathways. By combining classical parasitology, advanced molecular tools, and microbiome analysis within a One Health framework, this work advances understanding of the ecological and epidemiological complexity of intestinal microbial eukaryotes. The results underscore the urgent need for integrated, longitudinal, and seasonally stratified surveillance strategies to inform targeted public health interventions and to mitigate zoonotic risk in vulnerable rural communities.