Open Veterinary Journal, (2026), Vol. 16(3): -1635

Research Articlewww.eldaghayes.com

10.5455/OVJ.2026.v16.i3.20

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Microscopic and molecular identification of Toxocara cati in domestic and stray cats in Al Diwaniyah Province, Iraq

Noor Idan Jarad¹, Hadeel Hadi Al-Bayati^1*, Amal Hassan Al-shabbani² and Sarah Sadeq Shakir¹

¹Department of Microbiology, College of Veterinary Medicine, Al-Qadisiyah University, Al Diwaniyah, Iraq

²Department of Medical Clinic, College of Pharmacy, University of Al-Qadisiyah, Al Diwaniyah, Iraq

*Corresponding Author: Hadeel Hadi Al-Bayati. Department of Microbiology, College of Veterinary Medicine, Al-Qadisiyah University, Al Diwaniyah, Iraq. Email: hadeel.albayati [at] qu.edu.iq

Submitted: 15/09/2025 Revised: 30/01/2026 Accepted: 16/12/2025 Published: 31/03/2026

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ABSTRACT

Background: Because Toxocara spp. is considered one of the parasites that cause common diseases in cats, studies in Al-Diwaniyah Provicne, Iraq, are limited.

Aim: The goal of this study was to determine if there were any Toxocara parasites in domestic and stray cat and molecular identification of Toxocara investigate the impact of certain epidemiological variables on the infection rate, including sex, age, and months.

Methods: A total of 100 fecal samples were collected from cats from October 2024 to February 2025. Samples of both sexes, both kittens and adults.

Results: According to the microscopic analysis findings, 25 (25%) of the cats had Toxocara spp. infections. The target was internal transcribed spacer 1 of the 5.8S ribosomal RNA gene. The conventional polymerase chain reaction (PCR) assay was positive for 32% (32/100) of the samples. According to cat lifestyle, 42% of stray cats was infected, while 22% of domestic cats was infected, with a significant difference. Concerning age, kittens had a higher infection rate (45.8%) of Toxocara cati than adults (19.2%), with a highly significant difference. In comparison between both sex groups, males had a higher infection rate (45.6%) than females (20.3%), which was highly significant. Among the 5 months of the study, the infection rates were higher in February (60%), but decreased in October (10%), with a highly significant decrease. The sequencing results of DNA from feces of cats were verified by reference from the internal transcribed spacer 2 (size=300 bp) of Toxocara spp. Results of the phylogenetic tree were identified, and the strains in Iraq are identical to those in other countries. Toxocara cati in cats that is closely related to strains from Iran, France, Japan, and China was discovered.

Conclusion: There is a moderate prevalence of T. cati eggs in cats in Al-Diwaniyah. Kittens had a higher infection rate than adult cats, and male cats were more likely to be infected than female cats. Moreover, suggests that there are generally more stray cats than domestic cats. This led to increased infection rates in February. For Toxocara infection detection, PCR is the most efficient method due to its high sensitivity.

Keywords: Domestic and stray cats, Iraq, Microscopic examination, PCR assay, Toxocara cati.

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Introduction

Domestic and stray cats are the definitive hosts of Toxocara spp., which are considered zoonotic parasites of public health and veterinary concern (Alwan, 2014; Hadi, 2016; Jarad et al., 2019). This parasitic nematode can infect feline breeds. In addition to cats, it can infect other animals, including dogs, wild felids, and foxes (Fisher, 2003; Rostami et al., 2020). The life cycle of this pathogen begins when infected cats’ feces or contaminated soil contain infectious eggs that can be consumed. Every day, female worms deposit hundreds of millions of eggs, which develop into up to 10 cm long adult worms (Peter et al., 2011; Monteiro et al., 2016). The main risk factors for toxocariasis include owning cats as pets, playing and petting them, youngsters engaging in geophagy, allowing dogs and cats to roam freely in public parks and countryside, and eating unwashed vegetables without following sanitary regulations (Lucio-Forster et al., 2016). Clinically, microscopy and molecular methods can be used to identify the infection in cats. Abdominal pain, vomiting, diarrhea, weight loss, and physical condition loss are all possible clinical indicators of Toxocara cati infection (Overgaauw and Nijsse, 2020). The physical traits of T. cati in feline breeds may be revealed by a microscopic examination, which may be checked for T. cati eggs from sick cats (Remesar et al., 2022). A molecular analysis could reveal details about the genetic composition of this parasite in domestic cats, which might involve amplification of specific genes from T. cati, such as the second internal transcribed spacer (ITS-2) region of ribosomal DNA (Hade, 2020; Fadhil et al., 2022). Between 20% and 40% of cats in Europe have endoparasites (Beugnet et al., 2014). Additionally, stray or free-ranging outdoor cats are more likely to have parasites than cats kept indoors, according to multiple studies. For example, compared with owned cats, stray cats in Greece had an 8.8-fold higher risk of contracting toxocariasis. Similarly, cats that lived outside had a 2.7-fold higher T. cati infection rate than those that stayed indoors (Chalkowski et al., 2019; Strube et al., 2019). As mentioned previously, some Iraqi cats frequently have T. cati infections. In Mosul, Al-Obaidi (2012) discovered a 40% frequency. In Baghdad, the frequency was 12.9% (Hadi and Faraj, 2014). This research was carried out using microscopic and molecular characterizations because there is currently no epidemiological study of this disease among household cats or molecular data about it in Al-Diwaniyah province as a whole.

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Materials and Methods

Collection of the samples

The research region was the Iraqi governorate of Al-Diwaniyah. The samples were collected between October 2024 and February 2025. We collected 100 fecal samples from both domestic and stray cats (50 samples from domestic cats and 50 from stray cats). Cat owners and veterinary facilities supplied the samples. The sample number, date, age, sex, and months were recorded on a plastic jar containing 6–8 g of cat feces. A portion of each sample is frozen for molecular analysis, while 3–5 g are taken for microscopic examination of the parasite.

Microscopic analysis

Microscopic analysis of feces is a common diagnostic technique for determining the presence of eggs or larvae in the gastrointestinal tract of animals, such as cats with T. cati infection. A direct fecal smear approach was employed to identify the parasite’s eggs (Urgel et al., 2019). One popular diagnostic technique for finding parasite eggs is the floating technique. This technique involves combining a small amount of waste with a flotation solution (Sheather’s solution) in a test tube. Because the flotation method has a higher specific gravity, eggs float to the top. After a few minutes, coverslips are taken and put on microscope slides (Jarad et al., 2019; Albayati et al., 2020; Jarad et al., 2025; Resan and Jarad, 2025).

Molecular method

Polymerase chain reaction (PCR) and DNA isolation

The QIAmp DNA Stool Mini kit (Qiagen) was used to recover egg DNA from Toxocara excrement in accordance with the manufacturer’s recommendations for the PCR: The samples (the sediment mentioned above) were digested by proteinase K at 70°C for 30 minutes after being heated to 95°C for 30 minutes in Buffer ASL. The final DNA dilution was prepared in 80 µl of elution buffer and kept at −20°C before use. A spectrophotometer (Thermoscientific Nanodrop-ND 2000) was used to measure the amount of DNA in each sample.

Using gel electrophoresis

Agarose gel electrophoresis was used to analyze the PCR results in the following steps: 1. After dissolving 1.5% (1.5 g) of agarose powder in 100 ml of TBE buffer, the mixture was microwaved for 2 minutes to reach 95°C before being allowed to cool to 60°. 2. The melted agarose was then gently shaken to mix with 30 µl of ethidium bromide. When the comb was properly positioned, it was placed in the gel tray and left to set at room temperature for 20 minutes. At the end of the third solidification step, after carefully removing the combination from the tray, the gel tray was placed in the electrophoresis tank along with one TBE buffer. 4. Following the addition of 5 µl of PCR products to each well, an electric current was set for 1 hour at 100 V and 80 AM. 5. Finally, the gel documentation system was used to visualize the PCR results.

Analysis of the sequencing

DNA sequencing was performed on 10 PCR-positive Toxocara egg products. The High Pure PCR Clean-up microct (Roche, Germany) was then used to clean up the amplicons. Sentegen Biotechnology in Ankara performed nucleotide sequence analysis using databases and BLAST algorithms from the National Center for Biotechnology. The phylogenetic analysis was conducted using Mega software (Tamura et al., 2013). Drawing from the Kimura 2-parameter model of the software package, the tree was built using the neighbor-joining method (Saitou and Nei, 1987) in the software package. Bootstrap resampling was computed using 1,000 pseudo replicates and random seeds (Felsenstein, 1985).

Statistics analysis

Multiple evaluations of effectors were carried out using Chi–square (x²). Significant analyses were decided if p ≤ 0.05 (Al-Rawi, 2000).

Ethical approval

The protocol for this Research was approved by the University of Al-Qadisiyah, College of Veterinary Medicine, Department of Microbiology, to collect the necessary clinical samples. All participants were given formal consent. No. 3945 in 23-9-2025.

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Results

Microscopic examination results

The results of the concentration methods used in the present study revealed that the overall prevalence was 25 (25%) of 100 fecal samples tested. Light microscopy revealed that the eggs of T. cati were spherical to ovoid, dark brown in hue, and had thick pitted shells, as shown in Figure 1.

The molecular study findings are as follows

PCR of 100 fecal samples. The T. cati infection rate was 32% (32/100) in cats positive for internal transcribed spacer 1 (ITS1) 5.8S ribosomal RNA gene.

Toxocara cati infection rate according to lifestyle as determined by PCR

Domestic cats had a 22% infection rate, whereas stray cats had a 42% infection rate, with significant differences (Table 1, Fig. 2).

Fig. 2. Agarose gel electrophoresis with 1.5% agarose shows positive Toxocara cati amplicons (1–10) that target a 300-bp portion of the ITS2. NC stands for negative control when DNA was substituted with H₂O. M is a South Korean genedx molecular marker (100–1,500 bp).

PCR infection rate of T. cati according to age

The results showed that the infection rate in kittens (45.8%) was higher than that in adults (19.2%). With highly significant, as shown in Table 2.

Toxocara cati infection rate according to sex as determined by PCR

Toxocara cati was tested in 46 male and 54 female cat fecal samples. Of these, 21 male cats (45.6%) and 11 female cats (20.3%) had confirmed T. cati infections, with a highly significant difference (Table 3).

Toxocara cati infection rate per months as determined by PCR

Table 4 shows the monthly analysis results for every month from October 2024 to February 2025. The maximum infection rate of (60%) in cats was documented in February, and the lowest infection prevalence of (10%) was observed in cats in October. With a highly significant Table 5 NCBI-BLAST homology sequence identity (%) of local T. cati with deposited accession numbers (PV830596, PV830597, PV830598, PV830599, PV830600, PV830601, PV830602, PV830603, PV830604, and PV830605) and other sequences (Figs. 3 and 4).

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Discussion

Microscopic study

The study’s findings demonstrated that when 100 cat fecal samples were examined to determine the overall infection rate, the rate of T. cati infection in cats was 25%. Round to ovoid eggs are consistent with Fahrion et al. (2011), and they have a thick alveolar capsule and a dark brown granular material. Like a golf ball, the eggs are subspherical and have distinctly pitted surfaces. The results of Uga et al. (2000) are consistent with the fact that the use of microscope examination in this investigation did not yield an absolute detection result. Toxocara spp. eggs can be difficult to differentiate using traditional methods based on morphological characteristics (Oguz et al., 2018). For precise identification and diagnosis of Toxocara spp., PCR-based methods have been developed and used due to their high sensitivity, specificity, speed, and usability (Jarosz et al., 2021).

Molecular study

According to (Zangana and Erdeni, 2016), feces have contaminated the environment as a result of poor hygiene habits, cats roaming freely, inadequate veterinary care, carcasses, and animal offal, which could be the reason for the rate of 32% T. cati in cats by PCR in Al-Diwaniyah province, one important source of infections is soil pollution. PCR-restriction fragment length polymorphism, and real-time PCR are a few PCR-based molecular examinations that have been used to differentiate Toxocara spp. eggs (Durant et al., 2012; Öge et al., 2019a,b). Molecular tests are employed in studies on the lifecycles, systematics, and diagnostics of ascaridoid nematodes (Jarosz et al., 2021). In this study, DNA isolated from cat fecal samples was used to amplify the internal transcribed spacer 2 (ITS2) gene using a conventional PCR technique. The first and/or second ITS of ribosomal DNA (rDNA) have been demonstrated to be genetic markers for molecular systematic studies of different parasite groups (Xie et al., 2020). PCR methods based on the ITS1 and ITS2 regions of rDNA have been utilized in a number of studies to differentiate eggs of Toxocara spp., which share a common morphology (Sarani et al., 2022). A real-time PCR (qPCR) technique that targets the ITS sequences was employed to detect the species of Toxocara infection (Durant et al., 2012). In this work, they claimed that the newly created qPCR test may be considered a helpful instrument for identifying T. cati eggs in fecal samples. The 8.5S rRNA and ITS2 gene for fecal samples of cats were used in PCR to identify Toxocara spp. recovered from 100 fecal samples of cats in the city of Al-Diwaniyah (Fahrion et al., 2011). This study demonstrated that a PCR technique employing the ITS2 sequences as genetic markers offers a practical means of identifying and diagnosing T. cati. Which is accurately and unambiguously identified using this method. According to the findings of this investigation, PCR is the most effective means of identifying Toxocara spp. in cats, which is consistent with the signs of (Phoosangwalthong et al., 2022). Geographic variation and detection techniques may have contributed to the prevalence rate of T. cati in these studies. When talking about «prevalence,» it is important to keep in mind that various studies may yield different conclusions depending on a number of variables, such as sample size and sample type (corridors or necrophilia), as well as other statistical and epidemiological elements. Care should be taken while comparing and contrasting the results of several studies. The prevalence rates of T. cati in different regions and countries are reviewed here to show the global prevalence of this parasite. A noteworthy relationship was observed between infection rate and lifestyle in cats. The signs were corroborated by reports that stray cats were more likely to be infected with Toxocara spp. than dogs in Sulaimani province, Iraq, Kirkuk province (Northern Iraq) (Rashid et al., 2022). According to the findings, younger cats were more likely to be infected than older cats. According to research, cats under a year old have higher infection rates in the US, Mexico, the Caribbean, and Central America, whereas younger cats in Canada had a higher infection rate (Jenkins, 2020). The infection rates were highest during the colder months of February and lowest throughout October. Summer temperatures may reduce the chances of parasite survival; during the cooler months, the soil’s moisture content allows parasite eggs to hatch into contagious larvae (Al-Azizz, 2005) stated that a considerable percentage of diseases were found in Basrah throughout the winter. (Hade et al., 2018) stated that notable infection rates were observed in Baghdad in February and March.

Sequencing results

Revealed that 10 T. cati isolates (PV830596, PV830597, PV830598, PV830599, PV830600, PV830601, PV830602, PV830603, PV830604, and PV830605) were intimately associated with the NCBI-Blast T. cati isolate (PV830596), which was 100% identified in Iran (LC700102). Additionally, the T. cati isolate (PV830597) had a tight connection to the NCBI-Blast T. cati isolate (MT341304) in France, with 100% identification. We determined the 100% identification of PV830600, which has a close relationship to the NCBI-Blast T. cati isolate from Japan (AB110033). Additionally, isolate (PV830605) maintains close ties with the NCBI-Blast T. cati isolate from China (KY003067). However, other isolates (PV830598) were 99.62% related to Luxembourg (MT341312), (PV830601) were 99.62% related to Uzbekistan (PV366859), (PV830602) had a tight connection to India’s NCBI-Blast T. cati of (HQ389346), and (PV830604) was linked to (MT341304) in France. The results of this study are consistent with those of earlier research that used nuclear gene sequences to investigate genetic variances between Toxocara canis and T. cati and other Ascaridoid nematodes around the world, indicating the presence of evolutionary linkages and genetic variation (Hade et al., 2018; Xie et al., 2020; Fava et al., 2020; Alfatly and Jarad, 2025). Because these countries are close to one another and share a single geographic line, paratenic hosts—such as birds—can migrate between them. This could explain the extremely recognizable cluster inside the same nodule and their close interaction.

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Conclusion

The province of Al-Diwaniyah has a moderate number of T. cati eggs in cats. Male cats had a higher infection rate than female cats, and kittens had a higher infection rate than adult cats. The infection rate was also higher among stray cats than among household cats. Infection rates were therefore higher in January and February. The sensitivity of PCR makes it the most effective technique for detecting Toxocara infections.

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Acknowledgments

The author expresses gratitude to the College of Veterinary Medicine and College of Pharmacy, University of Al-Qadisiyah, Iraq, for their valuable technical assistance.

Conflict of interest

The authors declare no conflict of interest.

Funding

This study did not receive any funding. Each author made a self-supporting contribution to the overall work.

Authors’ contributions

Hadeel Hadi Albayati, Noor Idan Jarad, and Amal Hassan Al-Shabbani were involved in the conceptualization; Hadeel Hadi Albayati and Amal Hassan Al-Shabbani conducted the methodology; Noor Idan Jarad and Sarah Sadeq Shakir conducted the formal analysis; Noor Idan Jarad and Amal Hassan Al-Shabbani were in charge of the investigation, data curation, and study validation; and Hadeel Hadi Albayati and Sarah Sadeq Shakir worked on the writing and reviewing. Hadeel Hadi Albayati managed the project. All authors approved the final draft of the work.

Data availability

All data were provided in the manuscript.

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Fig. 1. T. cati eggs were found using light microscopy at 40x magnification.

Table 1. Toxocara cati infection prevalence in both stray and domestic cats.

Table 2. Age-related changes in cats’ Toxocarasis infection rates.

Table 3. Sex’s impact on cats’ Toxocara cati infection rates.

Table 4. Impact of monthly percentage on infection rate.

Table 5. Using deposited accession numbers, the NCBI-BLAST homology sequence identity (%) in local Toxocara cati.

Fig. 4. Multiple sequence alignment of the identified sequences showing the similarity and differences of the sequences compared with the global isolates.

Fig. 3. Evolutionary tree analysis of Toxocara cati using the maximum likelihood method and inferred by Tamura-Nei model. A matrix of pairwise distances calculated using the Tamura-Nei model was used to automatically build the beginning tree or trees for the heuristic search using the Neighbor-Join and BioNJ algorithms. Following that, we chose the topology with the highest log likelihood. In this investigation, 17 nucleotide sequences were analyzed. The final dataset included 260 locations. MEG11 was used to perform evolutionary analysis.