JAHP_13_s1_187-195

Introduction

The protozoa of the genus Cystoisospora cause an intestinal parasitic illness known as cystoisosporiasis (previously referred to as isosporiasis). This parasite infects both mammals and humans and is found worldwide, although it is most prevalent in the tropics and subtropics (Dubey, 2018). Coccidia are a kind of protozoan parasite that infects the intestines and is common in animals, people, and pets (Imran and Alsayeqh, 2022). Differences in oocyst shape, host specialization, and developmental phases make the genus Cystoisospora difficult to understand (Barta et al., 2005; Raza et al., 2018). Puppies younger than six months and dogs with reduced resistance systems are more likely to get diarrhoea caused by Cystoisospora species (Buehl et al., 2006). Contaminated intermediate hosts, such as rats and other animals, can spread the disease through the faecal-oral pathway or by consuming contaminated tissues. In the first, subclinical stage, no symptoms are present; in the second, clinical stage, symptoms such as oocyst shedding, anorexia, fever, watery diarrhoea, vomiting, dehydration, gastrointestinal haemorrhage, and abdominal pain are noticed (Papazahariadou et al., 2007; Lappin, 2010; Mohsen et al., 2024). In severe cases, respiratory distress, neurological symptoms, and even mortality can occur. Recovery may lead to the development of varying levels of immunity. In dogs, the survival of oocysts and their environmental persistence may contribute to ongoing transmission (Liberato et al., 2018). Dogs serve as hosts for four recognized Cystoisospora species. Among them, C. canis produces large oocysts (>33 µm), which can be distinguished from the smaller and structurally similar C. ohioensis, C. neorivolta, and C. burrowsi (<30 µm) (Lindsay et al., 1997a). These three are commonly grouped under the term C. ohioensis-like due to the challenges of morphological differentiation. Recent molecular tools, such as PCR, sequencing, and RFLP analysis, have enabled the more accurate distinction of these species (Samarasinghe et al., 2008; Matsubayashi et al., 2011; He et al., 2012).

It was initially believed that Cystoisospora spp. belonged to the Eimeriidae family. However, phylogenetic studies have shown that they are closer to the Sarcocystidae, which include coccidia that produce tissue cysts, such as Toxoplasma gondii and Neospora caninum (Barta et al., 2005; Carreno et al., 1998; Franzen et al., 2000).

Cystoisospora spp. is common in dogs worldwide, according to many epidemiological studies; 4.9% of dogs in South Korea tested positive (Liyanagama et al., 2024), 7.3% in Germany (Murnik et al., 2023), 24.6% in Spain (Ortuño et al., 2014), and 10.4% in Canada (Villeneuve et al., 2015). Microscopic and molecular studies revealed a canine infection incidence of 9.5% in Basrah, Iraq (Jassim et al., 2024), in contrast to previous research from Diwaniyah, Iraq, which indicated a prevalence rate of 17.3% by PCR and 9% by microscopic inspection (Jarad et al., 2025). The microscopic study revealed a 17.33% infection rate of Cystoisospora spp. in Babylon, Iraq (Abd Alhamza and Al-Hasnawy, 2025).

As they scavenge, dogs in urban areas of Iraq frequently encounter polluted surfaces since they are allowed to wander freely in neighbourhoods and households. This environmental exposure may increase the risk of parasite transmission through faeces, soil, and water. Although zoonotic parasites have received some attention, infections with Cystoisospora remain underreported. Although previous studies in Babylon province investigated some coccidian protozoa, such as Cryptosporidium spp, which revealed the infection rate was 22% in dogs (Alasady and Al-Hasnawy, 2024), Sarcocystis spp. (88.33%) in cattle (Kadhim and Ameer, 2025).

Molecular techniques were not successful in identifying canine illnesses caused by Cystoisospora species in this province. Therefore, this study set out to determine the molecular characteristics and prevalence of Cystoisospora.

Materials and Methods

Samples collection

From October 2024 to February 2025, researchers in Babylon province gathered 150 faecal samples from dogs, both stray and domestic, spanning a range of ages and sexes. Each sample was placed in a clean, labelled plastic container with a tightly sealed lid, and metadata such as age, sex, and faecal consistency were recorded at the time of collection. The samples were promptly placed in a cool box and sent to the Parasitology Laboratory, where they were stored at 4°C until the DNA extraction process could commence.

Molecular diagnosis

DNA extraction

After flotation purification, genomic DNA was extracted from 150 faecal samples following the directions of the Easy Pure® Stool Genomic DNA Kit (TransGen Biotech, China) (Abed et al., 2024).

Primer preparation and PCR amplification

For the molecular identification of Cystoisospora spp., an internal transcribed spacer-targeting primer pair (ITS-1) in the 18S rRNA gene region (450 bp) was used, as defined by Samarasinghe et al. (2008). The lyophilized primers were initially dissolved in nuclease-free ddH₂O to create a 100 pmol/µL standard solution. A working explanation of 10 pmol/µL was prepared by diluting 10 µL of the stock in 90 µL of ddH₂O.

PCR reactions were performed using a 25 μL final volume that included 12.5 μL of GoTaq® Green Master Mix (Promega, USA), one microlitre each of forward and reverse primers, 2 microlitres of template DNA at a concentration of 10 pmol/μL, and enough nuclease-free water to make up

Table 1: The sequence of primers used in this study.

Primer	Sequence	Primer sequence 5'- - 3'	Tm (°C)	GC%	Size of product (bp)	Source
ITS	F	GATCATTCACACGTGGC CCTTG	63. 7	55		(Samara et al., 2008)
ITS	R	GACGACGTCCAAATCCA CAGAGC	65. 1	57	450 bp	(Samara et al., 2008)

the whole volume. In a heat cycler, the PCR amplification was carried out using Applied Biosystems™ ProFlex™ PCR System (Fisher Scientific/ USA), subjected to the following cycling conditions: A three-minute denaturation at 94°C, forty cycles of two-minute denaturation, one-minute annealing, two-minute extension, and seven-minute extension, as indicated in Table 2.

Electrophoresis on 1.5% agarose gels was used to resolve PCR products, which were stained with RedSafe™ dye (Mebep Bio Science, China), and then viewed under UV light using a microscope. To measure the molecular size, a 100 bp DNA ladder (lane M) was used. The presence of a ~450 bp band was considered indicative of a positive result for Cystoisospora spp.

Table 2: The optimum condition of detection.

Phase	Tm (°C)	Time	No. of cycle
Initial Denaturation	94°C	3 min	1 cycle
Denaturation -2	94°C	2 min	40 cycles
Annealing	68°C	1 min
Extension-1	72°C	2 min
Extension -2	72°C	7 min.	1 cycle

DNA sequencing technique

The Cystoisospora species was determined using DNA sequencing, and the genotypes of thirteen local isolate products that tested positive for PCR were transferred to the MacroGene Company in Korea for further analysis. The entire sequence of the 18S rRNA gene was cast-off to confirm that all four isolates were C. canis (Jarad et al., 2025). We used BioEdit, version 7.2, in conjunction with the Basic Local Alignment Search Tool (BLAST) offered by the National Centre for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) to perform a homology search (Yun et al., 2023). To validate the findings, they were cross-referenced with information from the NCBI database and the ExPASy software (SIB Swiss Institute of Bioinformatics, Switzerland).

Phylogenetic tree analysis

To calculate evolutionary distances, a phylogenetic tree was constructed using the UPGMA technique (Sneath and Sokal, 1973). This is the ideal tree of C. canis, with a total branch length of 0.04032703. Here we can see the ideal C. ohioensis tree, where the total branch length is 7.22043733 (Tamura et al., 2004). The MEGA6 system was used to carry out evolutionary analyses (Tamura et al., 2013).

Statistical analysis

We expressed the results as a percentage. For the statistical analysis, we relied on the chi-square (χ²) test. Findings were considered statistically significant when the p-value was less than or equal to 0.05, and highly significant when the p-value was less than 0.001.

Results

Conventional PCR

Ten per cent, or fifteen out of one hundred fifty cases, had positive results for 18S rRNA gene PCR amplification, according to the molecular diagnostic data (Figure 1). A higher number of oocysts was found in the faecal samples of males (10.46%) compared to females (9.37%), with no significant difference (Table 3, Figure 2).

Table 3: Infection rate of Cystoisospora spp. in dogs according to the sex of dogs.

Sex	Examined sample	Infected samples	Infection rate
Male	86	9	10.46 %
Female	64	6	9.37%
Total	150	15	10 %
Chi-square (x2)	4.200
p-value	0.122

Puppies less than six months of age had the highest infection rate in this study’s PCR analysis, at 24.44% (11 out of 45). In contrast, the lowest infection degree was observed in dogs older than one year, with only 1 out of 45 animals testing positive (2.22%). These differences were statistically significant (p ≤ 0.01), as shown in Table 4 and Figure 3.

Table 4: Infection rate of Cystoisospora spp. in dogs according to age groups.

Age (months)	Examined sample	Infected samples	Infection rate
Less than 6 months	45	11	24.44%
6-12 months	60	3	5%
More than 1 year	45	1	2.22%
Total	150	15	10%
Chi-square (ꭓ2)	17.467
p-value	0.001**

*Highly significant difference at (P≤0.05). ** refer to significant difference at (p≤0.01).

DNA sequence analysis of Cystoisospora spp.

Thirteen canine faecal sample isolates from Babylon province were genetically sequenced and characterized using 18S ribosomal RNA gene partial sequencing in order to study and identify the species of genus Cystoisospora that

Table 5: DNA sequence analysis of Cystoisospora spp. according to 18S ribosomal RNA gene.

Identities	Sequence ID with comparison	Source	Nucleotide	Location	Type of substitution	No. of sample
99%	ID: OP837796.1	C. ohioensis	G\A	187	Transition	1
99%	ID: OP837796.1	C. ohioensis	C\G	246	Transvertion	1
99%	ID: OP837796.1	C. ohioensis	A\T	282	Transvertion	2
			A\C	289	Transvertion
			T\C	300	Transition
99%	ID: OP837796.1	C. ohioensis	A\T	282	Transvertion	3
99%	ID: MH790139.1	Cystoisospora canis	C\A	968	Transvertion	4
99%	ID: MH790139.1	Cystoisospora canis	A\C	1217	Transvertion	4
99%	ID: MH790139.1	Cystoisospora canis	C\A	968	Transvertion	5
			C\G	1086	Transvertion
			A\G	1087	Transition
99%	ID: MH790139.1	Cystoisospora canis	C\A	968	Transvertion	6
99%	ID: MH790139.1	Cystoisospora canis	C\A	1204	Transvertion	6
99%	ID: MH790139.1	Cystoisospora canis	C\A	968	Transvertion	7
			A\T	1078	Transvertion
			C\T	1245	Transition
99%	ID: MH790139.1	C. canis	C\A	968	Transvertion	8
99%	ID: MH790139.1	C. canis	C\A	968	Transvertion	9
99%	ID: MH790139.1	Cystoisospora canis	C\A	968	Transvertion	10
99%	ID: MH790139.1	Cystoisospora canis	A\G	1144	Transition	10
99%	ID: MH790139.1	Cystoisospora canis	C\A	968	Transvertion	11
99%	ID: MH790139.1	Cystoisospora canis	G\T	1146	Transvertion	11
99%	ID: MH790139.1	C. canis	T\G	920	Transvertion	12
99%	ID: MH790139.1	C. canis	C\A	968	Transvertion	12
99%	ID: MH790139.1	Cystoisospora canis	T\G	920	Transvertion	13
			C\A	968	Transvertion
			A\T	1078	Transvertion
			T\G	1210	Transvertion

had been identified by traditional PCR. The results showed that certain C. canis (10/13, 76.92%) and C. ohioensis (3/13, 23.07%) were present. Iso lates of Cystoisospora spp. were entered into the National Centre for Biotechnology Information’s GenBank database as accession numbers (ID: PV606919.1; ID: PV606920.1; ID: PV606921.1) and identified and registered for the specific species C. ohioensis. The sequences (ID: PV606922.1; ID: PV606923.1; ID: PV606924.1; ID: PV606925.1; ID: PV606926.1; ID: PV606927.1; ID: PV606928.1; ID: PV606929.1; ID: PV606930.1; ID: PV606931.1) were identified and registered for the specific species C. canis, expressed in Table 5.

Phylogenetic analysis of C. canis and C. ohioensis

The evolutionary history of C. canis and C. ohioensis was inferred using the UPGMA method (Sneath and Sokal, 1973), based on 18S ribosomal RNA gene sequences. To determine evolutionary distances, we employed the Maximum Composite Likelihood method (Tamura et al., 2004), which is based on the total number of nucleotide substitutions (or base changes) that occurred at each location. We used MEGA6 for all our analyses (Tamura et al., 2013), which included the first, second, third, and noncoding codon locations, and excluded any gaps or missing data.

For C. canis, the analysis included 16 nucleotide sequences with 350 aligned positions, yielding a best tree with a whole-branch distance of 0.04032703 (Figure 4). Isolates from Iraq shared 99% sequence similarity with strains from Canada and China (Table 6).

In comparison, the C. ohioensis phylogenetic tree was based on 10 sequences with 271 aligned positions, and a total branch length of 7.22043733 (Figure 5). Iraqi C. ohioensis isolates clustered closely with sequences from China, the USA, Australia, and South Korea, showing 91%–99% sequence similarity (Table 7).

Table 6: The NCBI-BLAST Homology Sequence Identity (%) between local C. canis in dog stool isolates and NCBI-BLAST deposited strains.

Source: Cystoisospora canis; 18S ribosomal RNA gene
Accession	Country	Isolation source	Date of registration	Compatibility
ID: MH790139.1	China	Dog	2018	99%
ID: KT184362.1	Canada	Dog	2015	99%
ID: KT184360.1	Canada	Dog	2015	99%
ID: KT184368.1	Canada	Dog	2015	99%
ID: KT184361.1	Canada	Dog	2015	99%
ID: KT184363.1	Canada	Dog	2015	99%

Table 7: The NCBI-BLAST homology sequence identity (%) between local Cystoisospora ohioensis in dog stool isolates and NCBI-BLAST deposited strains.

Source: Cystoisospora ohioensis; 5.8S ribosomal RNA gene
Accession	Country	Isolation source	Date of registration	Compatibility
ID: OP837796.1	China	dogs	2022	99%
ID: OP837793.1	China	dogs	2022	99%
ID: EU124688.1	Australia		2007	99%
ID: GU292307.1	USA	dogs	2009	99%
ID: GU292306.1	USA	dogs	2009	99%
ID: OM870403.1	Australia	fox	2022	93%
ID: 0Q473126.1	S. Korea		2023	91%

Discussion

This study is proposed as the first contribution to the knowledge of the attendance and prevalence of Cystoisospora species in dogs in the Babylon area, Iraq. The initial examination of faecal samples showed that most positive cases occurred in dogs with watery diarrhoea. Mitchell et al. (2007) reported that Cystoisospora infections in immunocompromised humans and dogs involve extra-intestinal stages linked to watery diarrhoea and weight loss. This supports the current findings, which indicate that cases of watery diarrhoea are associated with the highest infection rates in dogs.

Oocyst morphology has been the standard method for identifying Cystoisospora species to date. However, molecular confirmation using DNA analysis has been infrequently applied. As a result, the GenBank database contains limited 18S rRNA sequence data for Cystoisospora spp.

The current data revealed that the overall prevalence of Cystoisospora spp., as determined by conventional PCR, was 10%. This finding agreed with a study in Canada, which revealed the infection rate was 10.4% (Villeneuve et al., 2015), and it was also consistent with a study in Germany (7.3%) (Murnik et al., 2023). However, research conducted in South Korea found no such thing (Liyanagama et al., 2024), which reported an infection rate of Cystoisospora of 4.9%. Various geographical climates, host distribution, and other variables likely contributed to the disparity in prevalence rates of this parasite among nations or areas within a country (Confalonieri et al., 2014).

According to sex, the infection rate in dogs was higher in males compared to females, with no significant differences shown in the infection rate between males and females. This result was consistent with a study in the southern part of Iraq. Basrah, during which it was discovered that the prevalence of Cystoisospora infection was greater in male canines (6.6% vs. 6.3%) (Al-Jassim et al., 2017). However, it has issues with the findings of Jarad et al. (2025) in Diwaniyah city, Iraq, which revealed a higher contagion rate of C. canis in female dogs (22.34%) compared to male dogs (16.03%), as well as a variation in infection rates of Cystoisospora spp. When it comes to canines, this discrepancy may be due to variations in research sites, study periods, sample sizes, and diagnostic methods. Another possible factor is the existence of conditions that allow oocysts to survive for an extended period (Arsalan et al., 2006).

The PCR results revealed that the infection rate was highest in puppies under six months and lowest in dogs older than one year in this research. These results agreed with those of previous research that also considered animal age. Taking Germany as an example, Murnik et al. (2023) reported an infection rate of 14.0%, which exceeds the indicated prevalence. Villeneuve et al. (2015) found that canines less than one year old had the highest infection rate (12.6%). In France, Osman et al. (2015) found a significantly higher infection rate in puppies under one year (28.2%), in contrast to a significantly lower prevalence (15.6%) in dogs older than one year. In a similar vein, research in Diwaniyah city, Iraq, found that the infection rate was 38.02% in young pups and 8.52% in mature dogs, which aligns with the results of the present investigation (Jarad et al., 2025). The prevalence of parasite infection is significantly impacted by age. This finding aligns with Abere et al. (2013), who noted that the frequency of parasites was significantly linked to age. In addition, the same researcher reported that the parasite prevalence was higher in young dogs than in adults (Abere et al., 2013). Furthermore, Ahmed et al. (2014) noted that the prevalence of the infection was greater in pups compared to adults. This can be due to a lack of immunity during the subject’s youth, or it may be the result of a single or recurrent exposure (Ramírez-Barrios et al., 2004).

Regarding the DNA sequencing analysis, thirteen PCR products of the protozoan Cystoisospora isolated from dogs’ faeces were genetically analysed using partial 18S rRNA sequencing, identifying C. canis and C. ohioensis. Similar to previous studies, this investigation also recorded Cystoisospora spp. based on 18S rRNA sequencing, with the obtained sequences submitted to GenBank. In line with these efforts, in Diwaniyah city, Iraq, Jarad et al. (2025) reported a 19% prevalence of Cystoisospora spp. in stray dogs, as determined by microscopy. PCR and sequence analysis revealed parasite DNA in 56% of samples, showing 100% identity with C. canis strains from Canada. While in Basrah, Iraq, Jassim et al. (2024) recorded a 9.5% infection rate with C. canis in dogs, confirmed by PCR and DNA sequencing. The genetic results matched C. canis, confirming its presence and host specificity.

Globally, Cystoisospora spp. have been reported in dogs, with C. ohioensis identified as a common species. In Indonesia, Tokiwa et al. (2018) established the presence of C. ohioensis in dogs, and molecular analysis revealed 99.9% similarity with C. ohioensis based on the 18S rRNA gene. In Australia, Samarasinghe et al. (2008) conducted phylogenetic analysis of ITS1 orders from C. ohioensis -like oocysts. They found that they cluster within the family Sarcocystidae, rather than Eimeriidae, which supports their close evolutionary relationship to other tissue-cyst-forming coccidia, such as Toxoplasma and Neospora.

Based on the phylogenetic tree analysis. The Iraqi isolates of C. canis and C. ohioensis showed a high level of similarity to gene sequences deposited in the GenBank database. The C. canis isolates shared 99% sequence similarity with strains from Canada and China based on the 18S ribosomal RNA gene. Similarly, C. ohioensis isolates from Iraq showed close genetic relationships with strains from China, the USA, Australia, and South Korea, with sequence similarity ranging from 91% to 99%. According to the phylogenetic study, there were a few genetic variations between the Iraqi isolates and those from other nations. Differences in reference sequence length or sample origins, particularly in terms of geography, are potential causes of these variances. Several molecular methods were employed, including PCR and sequencing of partial or full-length gene fragments. According to Matsubayashi et al. (2011), Dogs infected with Cystoisospora species structurally and genetically identical to C. ohioensis have been identified through molecular and morphological investigations. Dog samples were genetically similar to those of raccoons, which may indicate that the two carnivorous hosts share a similar life cycle or mode of transmission.

Furthermore, the DNA sequences were analyzed using phylogenetic methods, which revealed that the raccoon and dog isolates belonged to the same clade as additional types of Cystoisospora, including C. ohioensis. According to these results, C. ohioensis is the most commonly shared type of canine infection. Using 18S rRNA and ITS-1 gene sequences, Cystoisospora isolates collected from dogs in Changchun, China, were subjected to a phylogenetic study. The results showed that one of the isolates, Chang Chun 2, was genetically identical to C. ohioensis, and another, Chang Chun 1, clustered separately but exhibited substantial genetic similarity to C. suis. These findings suggested either the presence of a unique, yet undiscovered species or the possibility of intra-species genetic variation (He et al., 2012).

Reiterating the dependability of molecular analysis, particularly when morphological traits alone are insufficient for species classification, our results align with comparable studies from Japan and Korea that employed the same target genes (18S rRNA and ITS-1). The data also show the importance of revising the genetic classification of Cystoisospora species found in dogs.

Tokiwa et al. (2018) initially documented an infection of Asian small-clawed otters (Aonyx cinereus) with Cystoisospora, a parasite often seen in cats and dogs; molecular investigation confirmed a high degree of genetic similarity. According to phylogenetic analysis, the otter isolate is closely related to C. rivolta, suggesting a strong evolutionary connection. Cystoisospora spp. may infect more carnivorous hosts than previously thought, according to this finding. This highlights the need for genetic surveillance and cross-species monitoring, especially in exotic animal populations. Previous investigations have indicated minimal genetic diversity among Cystoisospora spp., particularly C. canis and C. ohioensis. The substantial genetic similarity between the Iraqi isolates and those from other countries confirms this finding. It also shows potential for cross-species transmission.

Conclusion

Canines from Babylon province, Iraq, were tested for Cystoisospora species for the first time in this revolutionary investigation. Through phylogenetic analysis and sequencing of the 18S rRNA gene, two species were identified: C. canis and C. ohioensis, with C. canis being the more prevalent species. The study demonstrates that molecular methods offer greater accuracy than traditional morphological identification. Furthermore, the high genetic similarity between local and international isolates confirms the conserved nature of the 18S rRNA gene. These findings underscore the importance of incorporating molecular diagnostics into parasitological studies to enhance the understanding of parasite epidemiology, transmission, and species identification.

Acknowledgments

The authors are thankful to the Department of Parasitology at College of Veterinary Medicine /Al-Qasim Green University for providing the necessary facilities for this research.

Novelty Statement

This paper presented novel findings for the first time in Babylon province, Iraq where dogs were found to be infected by the protozoan Cystoisospora, as well as two species of this protozoan (C. canis and C. ohioensis) that have been identified in this region and recorded in the NCBI database. These findings are useful for authorities concerned in veterinary hospitals and private veterinary sectors in order to aware owners about this infection and its distribution among domestic and stray dogs of Babylon province.

Author’s Contribution

Mohammad H. Al-Hasnawy: ideas, design the study and analyzed the results. Abdullah Riyadh Abd Alhamza: data collection, analysis, draft writing, and interpretation. Both authors have read, reviewed, and approved the final manuscript.

Conflict of interest

The authors have declared that they have no conflict of interests in the publication of this research.

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