Serological and molecular investigation of Toxoplasma gondii in domestic cats in Dhaka City, Bangladesh

Abstract

Toxoplasma gondii is a globally prevalent zoonotic protozoan parasite with a complex life cycle, using felids as definitive hosts and various warm-blooded animals as intermediate hosts. This study aimed to assess the sero-molecular prevalence and risk factors of T. gondii infection in domestic cats in the Dhaka Metropolitan Area, Bangladesh. Blood and fecal samples from 184 cats were collected and analyzed using both enzyme-linked immunosorbent assay (ELISA) and conventional polymerase chain reaction (PCR) targeting the REP-529 gene. The seroprevalence of T. gondii was determined as 9.78% (18/184), while molecular analysis detected a slightly lower prevalence of 8.15% (15/184) among the sampled cats. Several host- and management-related factors were significantly associated with T. gondii infection in cats. ELISA positivity was significantly higher in non-dewormed cats, those with outdoor access, and those defecating outdoors, while PCR positivity was significantly associated with outdoor access and outdoor defecation. Multivariable logistic regression revealed that age, deworming status, and outdoor access were significant risk factors based on ELISA. In contrast, outdoor access alone was identified as a risk factor by PCR detection. The results indicate that T. gondii infection is common among domestic cats in Bangladesh, posing a potential risk of zoonotic transmission to humans. These findings highlight the importance of raising cat owners' awareness, enhancing public health education, and promoting preventive veterinary care to reduce the transmission of T. gondii between cats and humans.

1. Introduction

Rapid urbanization in Bangladesh, particularly in Dhaka City, has led to increased cat ownership, driven by modern lifestyles (Bhowmik et al., 2020). While cats offer psychological benefits, they can carry Toxoplasma gondii, the causative agent of toxoplasmosis, which is transmitted through contaminated food, water, soil, or undercooked meat and can pose serious health consequences, particularly in pregnant women and immunocompromised individuals (Kim et al., 2024). Globally, human seroprevalence of T. gondii ranges from 0.5% to 87.7%, with a mean of approximately 25.7% (Molan et al., 2019). Among felids, pooled prevalence estimates are around 35% for domestic cats and 59% for wild species (Montazeri et al., 2020). In Bangladesh, previous studies have reported variable seroprevalence, ranging from 16 to 39% in humans and 1.5–60% in cats (Hasan et al., 2024), highlighting a significant zoonotic burden. Most of these studies focused solely on serological detection, without incorporating molecular confirmation or detailed analysis of management and environmental risk factors. Comprehensive epidemiological studies of T. gondii are essential for understanding parasitic prevalence, identifying risk factors, and elucidating transmission dynamics, thereby guiding the development of effective control measures to reduce environmental contamination and protect both human and animal health.

This study aimed to determine the serological and molecular prevalence of T. gondii infection among domestic cats in the Dhaka Metropolitan Area, Bangladesh. IgG antibodies were detected using an enzyme-linked immunosorbent assay (ELISA), and parasite DNA was identified by polymerase chain reaction (PCR). Furthermore, potential risk factors associated with infection were evaluated based on serological and molecular findings.

2. Materials and methods

2.1. Sampling

This study, conducted from March 2023 to December 2024 in metropolitan areas of Dhaka (Fig. 1), focused on a cross-sectional survey of 115 households and 69 treatment-seeking cats at government and private pet clinics. Relevant epidemiological and managemental data, including sex, age, breed, vaccination status, diet, deworming history, disease history, outdoor access, defecation habits, health status, abortion history, and cohabitation, were recorded during sampling or through interviews with pet owners. Blood samples were collected from 184 cats via the cephalic or saphenous vein using a 3 ml syringe and a 23G butterfly needle. The samples were transferred to Vacutainer tubes without anticoagulant, labeled, and centrifuged at 3200 rpm for 10 min to separate serum. The extracted sera were transferred into sterile vials and stored at −20 °C until serological analysis. Fresh fecal samples from the same cats were collected immediately after defecation and placed into individually labeled sterile zipper bags. Samples were transported to the laboratory in an icebox, and an aliquot of each fecal sample was preserved in 2.5% potassium dichromate solution and stored at 4 °C until DNA extraction.

Fig. 1.

Location of Dhaka City, Bangladesh, showing the geographic distribution of the sampling site. Map generated using ArcGIS Pro 3.4.

2.2. Serological examination

Serum samples were tested for anti-T. gondii IgG antibody using a commercial ELISA kit (ID Screen® Toxoplasmosis Indirect Multi-species, ID.vet, France), which has a reported diagnostic specificity and sensitivity of 97.3% and 78.8%, respectively (Liyanage et al., 2021). Each sample was diluted 1:10 with the kit-provided buffer, and positive and negative controls were tested in duplicate. The optical density (OD) value of each well was measured at 450 nm using an ELISA microplate reader. Based on the measured OD values, the sample-to-positive (S/P) ratio was calculated for each sample using the following equation:

$$\mathrm{S}/\mathrm{P}\%=\frac{{\mathrm{OD}}_{\mathrm{S}}-{\mathrm{OD}}_{\mathrm{NC}}}{{\mathrm{OD}}_{\mathrm{PC}}-{\mathrm{OD}}_{\mathrm{NC}}}\times 100$$

Using the S/P ratio, samples were categorized as positive if the S/P % was 50% or higher, negative if it was 40% or lower, and borderline if between 40% and 50% (Xia et al., 2022).

2.3. Fecal DNA extraction and PCR amplification

Each preserved fecal sample was thoroughly washed with distilled water to remove residual potassium dichromate before DNA extraction. Genomic DNA was extracted using the FavorPrep™ Stool DNA Isolation Kit (Favorgen Biotech Corp., Taiwan) according to the manufacturer's instructions. PCR amplification was conducted targeting the REP-529 region of T. gondii using the primers (forward: 5′-AGGCGAGGGTGAGGATGA-3′, reverse: 5′-TCGTCTCGTCTGGATCGCAT-3′), as described by Can et al. (2014). Reactions were performed in 25 μl mixtures containing 12.5 μl 2× Rapid Taq Master Mix (Vazyme, China), 1 μl of each primer, 2 μl of DNA template, and 8.5 μl PCR-grade water. Cycling conditions were: 95 °C for 10 min; 35 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s; followed by 72 °C for 10 min. Each assay included positive (genomic DNA extracted from cat feces previously confirmed positive for T. gondii) and negative (nuclease-free water) controls and was conducted in triplicate. Amplicons were subjected to 1.5% agarose gel electrophoresis and visualized under UV illumination after ethidium bromide staining, targeting a 529 bp fragment.

2.4. Nucleotide sequencing and analysis

All amplified products were purified using Montage PCR filters (Millipore) and sequenced bidirectionally with the BigDye Terminator v3.1 kit on an ABI 3730 DNA Analyzer (Applied Biosystems, USA). Raw nucleotide sequences and chromatograms were inspected using EditSeq 5.0 and Chromas 2.4, respectively. Consensus sequences were generated through alignment and compared with homologous sequences in GenBank using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/blast/) to confirm T. gondii identity.

2.5. Statistical analysis

Epidemiological data were documented in Microsoft Excel and analyzed in R programming with the epitools and other relevant packages. Chi-squared tests and multivariable logistic regression analyses were performed to assess potential associations and correlations between T. gondii occurrence and different variables. Associations were considered statistically significant at p ≤ 0.05. Agreement between the ELISA and PCR tests was evaluated using Cohen's Kappa statistic, interpreted as: <0 = poor, 0–0.20 = slight, 0.21–0.40 = fair (regular), 0.41–0.60 = moderate, 0.61–0.80 = substantial, and 0.81–1.00 = almost perfect agreement. McNemar's test was applied to assess the significance of differences between the two methods.

3. Results

The overall seroprevalence of T. gondii in cats was 9.78% (18/184), and PCR targeting the REP-529 gene (Fig. 2) showed a positivity rate of 8.15% (15/184). The univariate analysis revealed several host and management factors that were significantly associated with T. gondii infection (Table 1). ELISA positivity was significantly higher in non-dewormed cats (14.9%, p = 0.017), cats with outdoor access (19.1%, p = 0.001), and those defecating outdoors (28.0%, p = 0.004). Similarly, PCR positivity was significantly associated with outdoor access (16.2%, p = 0.002) and outdoor defecation (24.0%, p = 0.004).

Fig. 2.

Gel image of the PCR products of the REP-529 gene from cat fecal samples. Lane M: 100-bp DNA ladder; Lane PC: Positive control (genomic DNA from previously confirmed T. gondii); Lane NC: Negative control (nuclease-free water); Lanes C177–C195: Representative fecal samples from different cats, showing successful amplification of the target sequence.

Table 1.

Analysis of potential risk factors associated with sero-molecular prevalence of T. gondii in domestic cats.

Variable	N	ELISA				PCR
		Prevalence (%)	95% CI	χ2	p-value	Prevalence (%)	95% CI	χ2	p-value
Sex
Male	96	11.5%	6.5–19.4	0.64	0.424	5.2%	2.2–11.6	2.32	0.127
Female	88	8.0%	3.9–15.5			11.4%	6.3–19.7
Age
>2 years	17	23.5%	9.6–47.3	5.08	0.079	11.8%	3.3–34.3	0.38	0.828
1–2 years	72	11.1%	5.7–20.4			8.3%	3.9–17.0
<1 year	95	6.3%	2.9–13.1			7.4%	3.6–14.4
Breed
Local	144	11.8%	7.5–18.1	3.07	0.080	7.6%	4.3–13.2	0.23	0.629
Cross	40	2.5%	0.4–12.9			10.0%	4.0–23.1
Vaccination status
Yes	88	5.7%	2.5–12.6	3.21	0.073	4.5%	1.8–11.1	2.93	0.087
No	96	13.5%	8.1–21.8			11.5%	6.5–19.4
Diet
Cat food	13	23.1%	8.2–50.3	4.97	0.174	15.4%	4.3–42.2	3.69	0.298
Raw animal organ	3	33.3%	6.1–79.2			33.3%	6.1–79.2
Mixed	54	7.4%	2.9–17.6			7.4%	2.9–17.6
Cooked meat	114	8.8%	4.8–15.4			7.0%	3.6–13.2
Deworming status
No	94	14.9%	9.1–23.5	5.69	0.017⁎	9.6%	5.1–17.2	0.52	0.471
Yes	90	4.4%	1.7–10.9			6.7%	3.1–13.8
Disease history
Yes	48	12.5%	5.9–24.7	0.543	0.461	8.3%	3.3–19.6	0.01	0.957
No	136	8.8%	5.1–14.8			8.1%	4.6–13.9
Outdoor access
Yes	68	19.1%	11.5–30.0	10.65	0.001⁎	16.2%	9.3–26.7	9.28	0.002⁎
No	116	4.3%	1.9–9.7			3.4%	1.3–8.5
Defecation place
Outside	25	28.0%	14.3–47.6	10.89	0.004⁎	24.0%	11.5–43.4	10.82	0.004⁎
House floor	16	6.3%	1.1–28.3			12.5%	3.5–36.0
Litter	143	7.0%	3.8–12.4			4.9%	2.4–9.8
Health status
Healthy	62	8.1%	3.5–17.5	0.31	0.576	4.8%	1.1–13.7	1.37	0.242
Sick	122	10.7%	6.3–17.4			9.8%	5.7–15.8
Cohabitation
Yes	49	16.3%	8.5–29.0	3.24	0.072	4.1%	1.1–13.7	1.48	0.224
No	135	7.4%	4.1–13.1			9.6%	5.7–15.8
Sample origin
Households	115	6.9%	3.27–13.66	1.987	0.158	6.0%	2.69–12.58	1.09	0.29
Clinics	69	14.49%	7.5–25.5			11.6%	5.49–22.11

N/A: not measured due to the presence of zero values.

N = sample size, χ² = chi-square value, CI = confidence interval.

^⁎Statistically significant.

Multivariable logistic regression analysis showed that cats older than 2 years had significantly higher odds of ELISA seropositivity compared to those under 1 year of age (OR = 10.45, 95% CI: 1.63–70.85, p = 0.013) (Table 2). Similarly, cats that had not been dewormed (OR = 5.53, 95% CI: 1.20–32.41, p = 0.040) and those with outdoor access (OR = 4.76, 95% CI: 1.20–22.35, p = 0.033) were at significantly higher risk. Regarding PCR-based detection, outdoor access remained a significant risk factor (OR = 4.76, 95% CI: 1.10–25.50, p = 0.046) (Table 2), whereas no other variables were statistically significant in the regression model. Female sex showed a borderline non-significant association with increased PCR positivity (OR = 4.17, 95% CI: 1.04–20.91, p = 0.058).

Table 2.

Multivariable logistic regression analysis of various factors associated with sero-molecular occurrence of T. gondii in domestic cats.

Variable	ELISA			PCR
	Odds ratio	95% CI	p-value	Odds ratio	95% CI	p-value
Sex
Male	1.32	0.39–4.64	0.653	Ref.
Female	Ref.			4.17	1.04–20.91	0.058
Age
>2 years	10.45	1.63–70.85	0.013⁎	5.14	0.51-44.63	0.135
1–2 years	1.53	0.40–5.97	0.530	1.43	0.34–6.15	0.619
<1 year	Ref.			Ref.
Breed
Local	2.87	0.45–56.51	0.346	Ref.
Cross	Ref.			2.59	0.50–13.07	0.240
Vaccination status
Yes	1.05	0.20–5.17	0.952	Ref.
No	Ref.			3.49	0.66–23.43	0.162
Diet
Cat food	3.69	0.44–29.45	0.212	Ref.
Raw animal organ	1.95	0.04–66.19	0.710	2.19	0.04–100.02	0.673
Cooked meat	1.15	0.25–6.09	0.864	0.15	0.01–1.61	0.094
Mixed	Ref.			0.44	0.04–5.35	0.497
Deworming status
No	5.53	1.20–32.41	0.040⁎	Ref.
Yes	Ref.			2.69	0.57–13.75	0.213
Disease history
Yes	1.59	0.38–6.16	0.507	1.29	0.20–6.71	0.770
No	Ref.			Ref.
Outdoor access
Yes	4.76	1.20–22.35	0.033⁎	4.76	1.10-25.50	0.046⁎
No	Ref.			Ref.
Defecation place
Outside	2.28	0.51–9.88	0.272	2.11	0.27–23.04	0.499
House floor	1.29	0.06–9.85	0.830	Ref.
Litter	Ref.			0.18	0.02–1.80	0.109
Health status
Healthy	1.13	0.16–7.01	0.900	2.12	0.33–17.05	0.447
Sick	Ref.			Ref.
Cohabitation
Yes	1.73	0.39–7.22	0.456	Ref.
No	Ref.			8.71	0.91–164.87	0.097
Sample origin
Households	Ref.			Ref.
Clinics	1.51	0.45–5.12	0.50	2.17	0.55–9.41	0.27

CI = confidence interval, Ref. = reference category.

^⁎Statistically significant.

Among the 184 samples tested, 18 (9.78%) were positive by ELISA, and 15 (8.15%) were positive by PCR. Of the ELISA-positive samples, 5 (5/18; 27.77%) were also positive by PCR, while 13 (13/18; 72.22%) were PCR-negative. In contrast, 10 of 166 ELISA-negative samples (6.02%) tested positive by PCR. The majority of samples (156/184; 84.78%) were negative in both assays (Table 3). The Cohen's Kappa statistic was 0.235 (SE = 0.1492; 95% CI: −0.057 to 0.527), indicating the regular fair agreement between the two tests. McNemar's test yielded a statistic of 0.174 (p = 0.677), suggesting no significant difference between them.

Table 3.

Comparison of ELISA and PCR detection methods of T. gondii in cats.

Tests	PCR Positive (%)	PCR Negative (%)	Total (%)
ELISA Positive (%)	5 (2.71)	13 (7.07)	18 (9.78)
ELISA Negative (%)	10 (5.43)	156 (84.78)	166 (90.21)
Total (%)	15 (8.15)	169 (91.85)	184 (100)

Sequence analysis of the REP-529 gene in 15 PCR-positive samples confirmed the identification of T. gondii and revealed limited genetic diversity. Thirteen isolates showed 100% identity with the reference sequence from bat placenta in Brazil (GenBank accession no. PQ682527), while two isolates exhibited a single-nucleotide polymorphism at position 398 (T → G).

4. Discussion

The global seroprevalence of T. gondii in domestic cats is estimated at ∼35% (Montazeri et al., 2020). In this study, a lower seroprevalence of 9.78% was observed, similar to reports from Japan (9.0%) and Thailand (11.0%) (Matsuu et al., 2017; Jittapalapong et al., 2007), but lower than in Iraq (16.3%) and China (19.9%) (Khayat, 2022; Zhou et al., 2021). The molecular prevalence targeting the REP-529 non-coding region, noted for its 10–100-fold higher sensitivity (Homan et al., 2000), was 8.15%, comparable to Iran (8.0%) (Mosallanejad et al., 2017), higher than South Korea (4.5%) (Jung et al., 2015), yet lower than Malaysia, China, and Indonesia (10.5–33.3%) (Tan et al., 2020; Song et al., 2020; Hanafiah et al., 2018). The low seroprevalence likely reflects the demographics of the study, which primarily included owned household cats with restricted outdoor access. Additionally, the ELISA's low sensitivity (78.8%) may have led to some false negatives, resulting in an underestimation of the true exposure rate despite its moderate specificity (97.3%). These factors, combined with improved management in urban Dhaka, may have contributed to lower antibody levels than the global average.

Analysis of host- and management-related risk factors identified several variables significantly associated with T. gondii infection in cats. Seroprevalence increased with age, with cats older than two years showing the highest rates, likely reflecting cumulative exposure and prolonged antibody persistence (Schäfer et al., 2024), although some studies reported no significant age-related differences, and PCR detection rates were similar across age groups (Lakhamsen et al., 2022; Suwancharoen et al., 2022). The age-related increase in seroprevalence may be attributable to cumulative environmental exposure in Dhaka's urban setting over time. Moreover, because ELISA detects persistent IgG antibodies, the higher positivity in older cats reflects a history of both past and current infections, which naturally exceeds the exposure window of younger animals. Deworming status was significantly associated with seropositivity, with non-dewormed cats exhibiting higher prevalence, possibly due to reduced parasite burdens in dewormed animals that otherwise could compromise host immunity (Abbas et al., 2021; Said, 2023), whereas PCR-based prevalence did not differ between dewormed and non-dewormed cats (Altner et al., 2025). Most administered dewormers were standard anthelmintics rather than specific antiprotozoals. Although specific data on the active compounds used were not consistently available, this variable was included to assess whether secondary parasitic infections influence host susceptibility to T. gondii; the correlation likely reflects either a compromised immune response due to co-infection or a proxy for the overall quality of veterinary management.

Outdoor access emerged as a major risk factor, with both serological and molecular analyses showing higher prevalence and logistic regression indicating a 4.76-fold increased risk. This suggests that predation on local rodents or birds remains a major transmission route for domestic cats in this region, even in metropolitan areas (Altner et al., 2025; Ubaid et al., 2025). Cats defecating outdoors also had higher infection rates, reflecting exposure to contaminated soil, water, and feces, though multivariable analysis indicated no independent effect (Jung et al., 2015; Afonso et al., 2008). Vaccination status and diet showed non-significant trends toward increased risk. Unvaccinated cats had higher seroprevalence and PCR positivity, potentially due to greater susceptibility to concurrent infections (Galván-Ramírez et al., 2022; Kokkinaki et al., 2023). Similarly, cats fed raw animal organs exhibited higher infection rates, reflecting ingestion of tissue cysts (Dunay et al., 2024; Kokkinaki et al., 2023). Health status, cohabitation with other cats, and history of illness were not significant determinants of infection, although sick or previously ill cats showed slightly higher seroprevalence, likely due to compromised immunity, with PCR detection remaining comparable across groups (Hanedan et al., 2023; Arruda et al., 2021).

A higher proportion of cats were seropositive for T. gondii IgG than PCR-positive for parasite DNA, consistent with previous studies (Majid et al., 2021; Galván-Ramírez et al., 2022; Schäfer et al., 2024). The fair (regular) agreement between the tests (Cohen's Kappa = 0.235) and the non-significant McNemar's test (p = 0.677) underscore their complementary roles. Seropositivity reflects both past and current exposure due to the persistence of antibodies, whereas PCR positivity indicates active or recent infection, as oocyst shedding is brief and may occasionally result from transient passage of ingested cysts (Chi et al., 2021; Poulle et al., 2016). Combining serological and molecular testing thus provides a more comprehensive understanding of T. gondii infection dynamics and epidemiology in domestic cats (Kim et al., 2024).

This study has some limitations that should be considered when interpreting the results. First, the moderate sensitivity (78.8%) of the ELISA kit may have led to an underestimation of true seroprevalence. Second, we could not identify the specific deworming compounds used due to owner recall, preventing a direct assessment of antiprotozoal efficacy. Third, the absence of concurrent coproparasitological testing for oocyst detection and corresponding clinical health data limits our ability to confirm active shedding or symptomatic disease. Finally, our sample was primarily composed of owned cats in metropolitan Dhaka, which may not represent the infection dynamics in stray or feral populations across more diverse geographic regions of Bangladesh.

5. Conclusion

Domestic cats in Dhaka City exhibit a moderate seroprevalence and molecular prevalence of T. gondii. Multivariable analysis identified age (>2 years), lack of deworming, and outdoor access as significant risk factors for seropositivity. For molecular detection, outdoor access was the sole significant risk factor. These findings suggest that although infection rates are not high, management practices, specifically environmental exposure and preventive care, play a critical role in transmission. Targeted public health education and improved veterinary management are recommended to reduce the risk of toxoplasmosis in both humans and animals in Bangladesh.

CRediT authorship contribution statement

Md. Farhan Hasan: Writing – original draft, Investigation, Formal analysis, Data curation. Ainun Nahar: Writing – original draft, Formal analysis, Data curation. Anas Bin Harun: Writing – review & editing, Writing – original draft, Visualization, Software, Investigation, Formal analysis, Data curation. Abdullah Al Bayazid: Resources, Investigation, Data curation, Writing – review & editing. Sourov Sutradhar: Software, Formal analysis, Investigation. Sabiha Zarin Tasnim Bristi: Investigation, Data curation. Basant Saud: Investigation, Data curation. Delower Hossain: Writing – review & editing, Supervision, Formal analysis. Md. Aminul Islam: Writing – review & editing, Supervision, Formal analysis. Md. Ataur Rahman: Writing – review & editing, Supervision, Formal analysis. Md Robiul Karim: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.

Consent to participate

After clearly explaining the research objectives to the cat and dog owners, written informed consents were obtained before data and sample collection.

Consent for publication

Not applicable.

Ethics approval

The Animal Research Ethics Committee (AREC) of Gazipur Agricultural University (GAU), Bangladesh, reviewed and approved the methodology of this study (reference: FVMAS/AREC/2023/35).

Funding

This research was partially funded by the University Grant Commission (UGC), Bangladesh. The funder had no role in the study design, data collection and analysis and decision to publish this manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All unique gene sequences of T. gondii from this study have been archived in the GenBank database of the National Center for Biotechnology Information (NCBI) under unique accession numbers (PX118530-PPX118531).