Intestinal Protozoa at the Human, Animal and Environment Interface in Rural Iraq

1. Introduction

Intestinal protozoan parasites are globally significant pathogens, causing considerable morbidity in humans and livestock, particularly in low- and middle-income countries (LMICs)[1]. These organisms, including Cryptosporidium spp., Giardia duodenalis, Entamoeba histolytica, and Enterocytozoon bieneusi, contribute substantially to diarrheal disease, malnutrition, and economic losses in affected communities [2,3]. Beyond their clinical and veterinary impact, these parasites also pose an economic burden due to livestock losses and reduced productivity [4,5].

Cryptosporidium spp. and Giardia duodenalis are known zoonotic agents, frequently transmitted between humans and animals via the fecal-oral route, often through contaminated water or food [6]. Entamoeba histolytica is one of the few invasive amoebae and remains a major cause of dysentery worldwide [7]. Meanwhile, Blastocystis, a genetically diverse organism whose pathogenicity remains controversial, has garnered increasing attention in recent years due to its high prevalence in both healthy and symptomatic individuals, and potential associations with a healthy gut microbiome [8]. Finally, E. bieneusi is an emerging microsporidian parasite found in various hosts, including humans, and is particularly problematic in immuno-compromised individuals [9].

While these eukaryotic microbes have been studied globally, investigations in Iraq remain limited, with most relying on microscopy-based methods [10]. Studies based on molecular methods are scarce, particularly for Blastocystis and E. bieneusi. Prior reports have documented these protozoa in humans and animals in Iraq, but knowledge on their epidemiology and diversity remains limited [11,12,13].

To bridge these gaps, the current study employed both microscopic and molecular diagnostic methods to investigate the occurrence, genetic diversity, environmental contamination and potential zoonotic transmission of intestinal protozoa between humans and small ruminants in a rural village in Iraq.

2. Materials and Methods

2.1. Ethics

The Ethics Committee (ACUC) at the University of Baghdad approved this study on 16-03-2022 under the project "One Health Approach—Iraq" (No.D.A.672).

2.2. Study Area

The study took place at Alissma village, which is located in the northeastern part of Iraq, near the border with Iran (Figure 1). Approximately 1000 individuals reside in this village. The nearest city (Mandli) is about 30 km away. The village is located in a semi-desert area with nearly no tree cover, devoid of ponds and lakes. Oil River runs through the village in the winter, but its water is unfit for drinking and is used solely for irrigation purposes. Groundwater wells and underground well water are typically used for drinking after filtration and purification. The village latrines are basic and located outside the household.

2.3. Sample Collection

The methodology used herein is graphically summarized (Figure 2). A total of 140 samples were collected between February 2022 and July 2022. Fifty of these were stool samples collected from humans. The participants did not have diarrhoea or other gastrointestinal symptoms and were not taking antibiotics at the time of sampling, except four participants who had colitis as diagnosed by a physician. During sample collection, questionnaires were administered, and information about age, gender, type of drinking water, antibiotics, pets, career, and chronic disease was recorded.

Moreover, 50 samples were collected from animals, namely from goats (n=15) and sheep (n=35). These animals grazed near the village and drank unfiltered well water. None of the animals had diarrhea at the time of sampling.

Additionally, 20 water samples were collected from sources used by both humans and animals in 5 ml plastic tubes. These included filtered drinking water (n=5), tap water (n=5), well water (n=5), and river water (n=5). Twenty soil samples were also collected. These included home garden soil (n=5), field soil (n=5), animal grazing field soil (n=5), and river edge soil (n=5). Five grams of soil were collected in plastic tubes after scraping five centimeters from the surface of the soil.

2.4. Microscopic Examination

All samples were examined microscopically using wet mount smears. Iodine staining was used for the human and animal fecal samples. Samples were also observed using the concentration method which entailed mixing the samples with a salt solution, and subsequently examining the concentrated top layer for parasitic forms.

2.5. DNA Extraction and Molecular Detection

For DNA extraction, 200 mg of each fecal and soil sample was used using the PureLink™ Microbiome Genomic DNA Purification Kit (Invitrogen) according to the manufacturer’s protocol. The extracted DNA was used for qPCR and nested PCR (Table 1).

This study screened samples for Blastocystis sp., Cryptosporidium spp., Entamoeba histolytica, Giardia duodenalis and E. bieneusi. The SSU rRNA gene was used to identify Cryptosporidium spp. and gp60 was used for Cryptosporidium subtyping. For Blastocystis, SSU rRNA was used; beta-giardin (bg) and triosephosphate isomerase (tpi) for Giardia; and internal transcribed spacer (ITS) for E. bieneusi. A positive and negative control were included in each PCR run. The reaction conditions differed according to parasite and genetic marker (Table 1). Probe-based qPCR was used to amplify a fragment of the SSU rRNA of G. duodenalis and E. histolytica. The positive PCR products were purified using a Thermo Scientific GeneJET Gel Extraction Kit (Thermo Fisher Scientific, CA, USA) according to the manufacturer’s protocol. Methodology used in this study was according to previous investigations [14,15,16].

2.6. Sequencing Analysis

The purified positive PCR amplicons were sequenced unidirectionally at Eurofins genomics (Cologne, Germany). The obtained raw reads were trimmed manually at both ends to remove ambiguous bases using SnapGeneViewer v.6.0.2. The acquired sequences were used as queries to perform BLAST against the NCBI database. The newly generated nucleotide sequences were submitted to GenBank under accession numbers PV521084, PV521085, PV521086, PV521087, PV504621, PV504622, PV504623, and PV504624.

3. Results

3.1. Microscopic Examination

The stool and environmental samples were initially examined using light microscopy. A total of 12% (6/50) of the human samples were positive for Cryptosporidium spp., 16% (8/50) for Blastocystis sp., and 10% (5/50) for G. duodenalis (Table 2, Figure 3). Moreover, 26% (13/50) of the animal samples were positive for Cryptosporidium spp., 78% (39/50) for Blastocystis sp. [4% (8/50) of the Blastocystis sp. were from goats and 70% (35/50) were from sheep] and 8% (4/50) for G. duodenalis from sheep. Regarding the soil samples, 5% (1/20) tested positive for Cryptosporidium spp., and 45% (9/20) for Blastocystis sp. Regarding the water samples, 15% (3/20) were positive for Cryptosporidium spp. and 5% (1/20) for Blastocystis sp.

Giardia duodenalis and Enterocytozoon spp. were not detected by microscopy in any of the water or soil samples.

3.2. Molecular Detection

Based on qPCR analysis targeting a fragment of the SSU rRNA gene of G. duodenalis, 30% (15/50) of human samples were positive, 14% (7/50) of animals (all positives were from sheep), and 5% (1/20) of soil samples. Nested PCR of longer bg and tpi gene fragments amplified one of the qPCR positive sheep samples, which belonged to assemblage A.

Using nested PCR, one human sample was positive for C. parvum (gp60 gene) and another for Blastocystis ST1. In animals, Cryptosporidium spp. was detected in 4% (2/50); one sheep sample was positive for Cryptosporidium ubiquitum (SSU rRNA gene) and another for Cryptosporidium parvum (SSU rRNA, gp60 genes). 8% (4/50) of animals were positive for Blastocystis (SSU rRNA gene), one with ST4, and three with ST10. Finally, 8% (4/50) of animals were positive for E. bieneusi, with three belonging to E. bieneusi genotype BEB6 and one to genotype COS-I.

All samples were negative for Entamoeba histolytica.

Mixed infections were identified in 8% (4/50) of human samples, when combining microscopy and molecular detection methods (Figure 4). This included two samples that tested positive for Giardia duodenalis by molecular detection and Blastocystis sp. by microscopy. One sample was positive for both Cryptosporidium spp. and G. duodenalis by molecular detection, while another sample was positive for Cryptosporidium spp., G. duodenalis, and Blastocystis sp., all detected by molecular methods (Figure 5).

Regarding animal samples, 34% (17/50) had co-infections. Twelve were infected with Cryptosporidium spp. and Blastocystis sp. by microscopy-based detection, five with G. duodenalis and Blastocystis sp., this included three samples positive for G. duodenalis and Blastocystis sp. microscopically, one sample testing positive for both parasites molecularly and lastly, one tested positive for mixed infection, both microscopically and molecularly. Moreover, two animal samples tested positive for Blastocystis sp. by microscopy and E. bieneusi by PCR (Table 3).

4. Discussion

This study provides insights into the occurrence and genetic diversity of intestinal protozoan parasites in a rural village in Iraq. It highlights the zoonotic potential and environmental presence of species such as Cryptosporidium spp., Giardia duodenalis, Blastocystis spp., and Enterocytozoon bieneusi. Small ruminants are economically important animals in Iraq and are reared primarily on small- and medium-scale herds. Despite this, reports on their intestinal organisms are relatively sparse [17,18]. Herein, the occurrence rate of Cryptosporidium spp. in animals is lower than in previous studies, likely due to the methodologies used or the population examined. The occurrence in sheep is much higher than in goats, matching previous findings in the country [18]. This can be attributed to the free-range nature of goats as opposed to sheep [19]. The detection of Cryptosporidium parvum in both humans and sheep suggests zoonotic transmission within this community. Meanwhile, detection of Cryptosporidium ubiquitum and E. bieneusi in sheep hints at livestock as potential reservoirs for environmental contamination and human infection.

The high prevalence of Blastocystis in both animals and humans align with previous reports [20,21,22]. There was a notable difference between the detection methods used, with microscopy-based detection identifying many more samples as positive, rather than molecular methods, whereby it was not possible to amplify the corresponding gene fragment. The presence of co-infections, many of which were confirmed microscopically, could be a confounding factor here. While it is not possible to compare these findings to other studies in Iraq, previous molecular studies in neighboring Iran showed variable Blastocystis occurrence rates, with one as low 5% [23,24,25,26]. As this is, to the best of our knowledge, the first molecular detection study of Blastocystis in the country, further studies are needed to shed light on the organism’s epidemiology. The detection of ST4 in sheep is intriguing, as this subtype is typically linked to rodents and only infrequently reported in livestock. This is among the first reports of ST4 in sheep in this region, and as such, it could stipulate new insights into subtype distribution in the Middle East.

Interestingly, Entamoeba histolytica was not detected in any sample. Nonetheless, Entamoeba spp. have been detected in both microscopic and molecular-based investigations in ruminants in the country [27,28]. This discrepancy may reflect the increased specificity and sensitivity of molecular methods used in the present study, especially under challenging field conditions that may compromise the detection of fragile organisms such as E. histolytica.

Given the scarcity of molecular epidemiological studies in Iraq, direct comparisons to previous work are limited. Nonetheless, our findings are in line with some earlier reports, such as the 34% Giardia detection rate by microscopy in humans observed by Al-Hasnawy and Idan [29].

While E. bieneusi infections have been reported in birds in Iraq [30], this study contributes new evidence of its presence in livestock, expanding the known host range in the region.

Several limitations should be acknowledged. One major challenge was preserving and storing samples in a remote field setting, where access to cold-chain infrastructure was limited. Inadequate preservation may have reduced the sensitivity of both microscopy and molecular assays, especially for rapidly degrading parasites. This could explain certain species' absence or low detection rates in specific sample types. Additionally, although PCR-based methods were employed, only a subset of positive samples yielded high-quality sequences, limiting the depth of genetic characterization. Environmental samples, in particular, may have contained PCR inhibitors or low DNA concentrations, affecting amplification success. Our findings underline the need for standardized operating procedures for parasite sampling, preservation, and analysis in resource-limited settings. The adoption of field-friendly preservatives compatible with molecular diagnostics, along with optimized DNA extraction protocols for complex environmental matrices, would enhance data quality and comparability [31]. Another limitation of this study is its cross-sectional design, which provides only a single time point and does not capture seasonal trends or temporal changes in infection dynamics. Longitudinal studies incorporating repeated sampling from both hosts and environments are needed to clarify transmission pathways, sources of reinfection, and possible seasonal patterns [32].

Despite including asymptomatic individuals and animals, future research should aim to expand the number of sampling sites, include larger sample sizes, and integrate clinical and immunological data. Such approaches would allow a better assessment of the pathogenic potential and health impacts of these parasites.

Looking ahead, the high prevalence of Blastocystis a common yet enigmatic member of the gut eukaryome presents a valuable opportunity to study its interaction with the bacterial microbiota and host immune system. Future studies should consider using 16s rRNA sequencing or shotgun metagenomics to explore microbe-parasite interactions, particularly in communities with frequent co-infections [33]. These approaches could inform new strategies for diagnostics, surveillance, and intervention.

5. Conclusions

In conclusion, this study enriches the limited molecular data on intestinal protozoan parasites in Iraq, offering a comprehensive view of their prevalence in humans, livestock, and the environment. The findings emphasize the importance of enhanced sampling protocols, environmental monitoring, and capacity building to improve parasite detection and control in rural, resource-limited settings.