Molecular detection and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis in cats and dogs from metropolitan areas of Bangladesh

Abstract

Cryptosporidium spp. and Giardia duodenalis are intestinal protozoan parasites of zoonotic concern that cause gastrointestinal diseases in humans and various animals, including cats and dogs. This study investigates the prevalence, risk factors, and genetic diversity of Cryptosporidium spp. and G. duodenalis in domestic cats and dogs in Bangladesh to assess their zoonotic potential. Fecal samples were collected from 197 cats and 120 dogs in Dhaka and Gazipur metropolitan areas. We performed nested PCR targeting the small subunit ribosomal RNA (SSU rRNA) gene for Cryptosporidium and the β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes for G. duodenalis, followed by nucleotide sequencing and analysis. The overall prevalence of Cryptosporidium was 8.1 % in cats and 4.2 % in dogs, whereas G. duodenalis was more common, detected in 29.9 % of cats and 25 % of dogs. Among examined variables, only sex and food types were significantly associated with G. duodenalis infection in dogs and cats, respectively. Molecular analysis identified three Cryptosporidium spp. in cats, including C. felis (81.3 %), C. baileyi (12.5 %), and C. canis (6.3 %), whereas 100 % of Cryptosporidium isolates from dogs were identified as C. canis. Multilocus genotyping of G. duodenalis revealed both host-adapted and zoonotic assemblages. Assemblage A predominated in both cats and dogs, followed by F and C in cats and C and D in dogs, with mixed infections observed in both hosts. The detection of zoonotic species and assemblages underscores the potential role of cats and dogs as reservoirs for human infection. These findings highlight the importance of monitoring intestinal protozoa in companion animals and promoting appropriate hygiene practices within a One Health framework.

Highlights

• •Giardia duodenalis showed a higher prevalence than Cryptosporidium in both host species.
• •Cats harbored C. felis, C. baileyi, and C. canis, while only C. canis was detected in dogs.
• •G. duodenalis assemblage A was predominant, with assemblages C, D, and F also detected.
• •Female sex in dogs and food type in cats were associated with G. duodenalis prevalence.
• •The detection of zoonotic assemblages highlights the One Health relevance of these infections.

1. Introduction

Cats and dogs play an increasingly important role in human societies by providing companionship, promoting physical activity, and supporting mental well-being (Martins et al., 2023). In rapidly urbanizing communities, including Bangladesh, pet ownership is rising among smaller households seeking emotional and psychological benefits (Mamun et al., 2024; Do Vale et al., 2021). However, close human–animal interactions also raise concerns about zoonotic disease transmission. Cats and dogs harbor numerous pathogens and are implicated in nearly 60 % of known zoonoses, which are transmitted directly or indirectly through contaminated food, water, and the environment (Salyer et al., 2017).

Among zoonotic parasites, Cryptosporidium spp. and Giardia duodenalis are of particular global significance, ranked sixth and eleventh among the most important food-borne parasites (Plutzer et al., 2018). Cryptosporidium comprises 48 valid species and more than 120 genotypes, of which 23 species and two genotypes infect humans (Zikmundová et al., 2025). Cryptosporidium hominis and C. parvum are the predominant species infecting humans, whereas C. felis (typically found in cats) and C. canis (commonly found in dogs) occasionally cause infections in humans (Bulumulla et al., 2025; Oladele et al., 2025). G. duodenalis is a species complex consisting of eight genetic assemblages (A–H). Assemblages A and B are zoonotic, while C/D and F are host-adapted to dogs and cats, respectively, though cross-species infections occur (Jańczak et al., 2025).

Both parasites are transmitted via the fecal–oral route, and their resistant cyst/oocyst stages enable persistence in the environment (Deng et al., 2023). Infections cause diarrhea, weight loss, and, in vulnerable hosts, chronic or life-threatening illness (Plutzer et al., 2018). Persistent infections impair growth and cognition in children (Seabolt et al., 2022). In pets, infections are often subclinical, allowing silent carriage and zoonotic transmission (Taghipour et al., 2020).

Globally, Cryptosporidium causes ∼118,000 deaths in total and ∼69,000 child deaths annually, with global disease burden of 7.37 million disability-adjusted life-years (Kyu et al., 2025). The average prevalence of Cryptosporidium is 2.9 % in cats and 4 % in dogs, while G. duodenalis occurs at approximately 2.5 % in both species (Bouzid et al., 2015; Barrera et al., 2024). In Bangladesh, the prevalence of Cryptosporidium in humans ranges from 1.4 % to 96 %, and G. duodenalis from 3.8 % to 94.2 %, depending on the detection methods used. Molecular genotyping studies have demonstrated that C. hominis is the predominant species infecting children in Bangladesh, accounting for 71.4 % to 91 % of cases, followed by C. parvum (7–28.6 %) (Karim et al., 2024; Hira et al., 2011). Extensive gp60 (60-kDa glycoprotein gene) genotyping in Bangladesh has revealed substantial genetic diversity, with multiple C. hominis subtype families, including Ia, Ib, and Id, and representative subtypes such as IaA18R3, IbA9G3, and IdA15G1 (Gilchrist et al., 2018; Karim et al., 2024). Notably, C. parvum subtype IIdA15G1 has been identified in both human and cattle populations in Bangladesh, indicating potential zoonotic transmission (Karim et al., 2024; Carey et al., 2023). Despite these extensive genotyping studies in humans, comparable molecular data on Cryptosporidium species and subtypes in cats and dogs, animals that commonly share households with or roam near humans, remain limited. Meanwhile, the prevalence of Cryptosporidium was reported at 8.3 % in cats and G. duodenalis at 42.6 % in dogs, based solely on microscopic examinations, without molecular characterization. (Mahmud et al., 2014; Nipa et al., 2022).

Despite their public health importance, these parasites remain underdiagnosed in Bangladesh due to reliance on conventional methods, hindering understanding of their epidemiology and zoonotic potential (Thompson and Ash, 2016). Given the increasing pet ownership and intensified human–animal interactions in urban settings, molecular investigations of Cryptosporidium spp. and G. duodenalis are essential. Therefore, this study aimed to investigate the prevalence, genetic diversity, and zoonotic potential of these parasites in domestic cats and dogs in Bangladesh using molecular methods.

2. Materials and methods

2.1. Sampling

The study was carried out in the metropolitan areas of Dhaka and Gazipur, Bangladesh, from July 2023 to June 2025 (Fig. 1). The minimum sample size was calculated using the formula n = Z²P(1 − P)/d² (Pourhoseingholi et al., 2013), with an estimated prevalence of 13 % for G. duodenalis in cats and 7 % in dogs, 5 % precision, and 95 % confidence level, yielding a minimum of 174 cats and 100 dogs, respectively. Finally, fecal samples were collected from 197 cats and 120 dogs. Among the study population, 164 cats (83.3 %) and 97 dogs (80.8 %) were from Dhaka City, and 33 cats (16.8 %) and 23 dogs (19.2) were from Gazipur City. Samples were obtained mainly from household pets receiving care at government veterinary hospitals or private pet clinics in Dhaka. Additional samples were collected from stray and sheltered dogs in Gazipur City. Fresh fecal samples were collected from the top of the fecal mass immediately after defecation and were placed in sterile, labeled zipper bags. The samples were then transported in ice box to the Zoonosis Research Laboratory, Gazipur Agricultural University (GAU), Bangladesh, where they were preserved in 2.5 % (w/v) potassium dichromate at 4 °C for further experiment.

Fig. 1.

Map of the study areas.

Epidemiological data were collected using a structured, pretested questionnaire covering feline and canine demographic characteristics (age, sex, and breed), health-related status (deworming and vaccine status), and managemental and environmental factors (food type, water source, outdoor access, hunting status, and cohabitation). Data were collected through face-to-face interviews with pet owners. Among the 197 cats examined, slightly more than half were male (52.8 %), and females accounted for 47.2 %. The majority were younger than 1 year (53.8 %), followed by 1–2 years (37.6 %) and >2 years (8.6 %). Most cats were local breeds (77.2 %), while crossbreeds comprised 22.8 %. In the 120 dogs studied, males were more common (58.3 %) than females (41.7 %). The largest age group was 1–2 years (47.5 %), followed by >2 years (25.8 %) and <1 year (26.7 %). Unlike cats, most dogs were crossbreeds (53.3 %), while local breeds represented 46.7 %.

2.2. DNA extraction and PCR amplification

Approximately 200 mg of each preserved fecal sample was thoroughly washed with distilled water to remove residual potassium dichromate prior to DNA extraction. Genomic DNA was extracted using the FavorPrep™ Stool DNA Isolation Kit (Favorgen Biotech Corp., Taiwan) according to the manufacturer's instructions, and the eluted DNA was stored at −20 °C until use in PCR analyses. Nested PCR amplification of the Cryptosporidium small subunit ribosomal (SSU rRNA) gene was performed using KOD-Plus-High Fidelity DNA Polymerase (Toyobo, Japan) (Xiao et al., 1999), while G. duodenalis loci were amplified with TaKaRa Taq (Toyobo, Japan) for the β-giardin (bg) gene (Lalle et al., 2005) and 2× Rapid Taq Master Mix (Nanjing Vazyme Biotech Co., Ltd., China) for the glutamate dehydrogenase (gdh) (Appelbee et al., 2005) and triosephosphate isomerase (tpi) genes (Sulaiman et al., 2003). Amplifications were performed using an Applied Biosystems Thermal Cycler v2.09. Each PCR run included a positive control for Cryptosporidium or G. duodenalis DNA from calf feces and a negative control with nuclease-free water to validate assay performance, and all samples were analyzed in triplicate to ensure reproducibility.

2.3. Nucleotide sequencing

All the secondary PCR products were sequenced in both forward and reverse directions by Sangon Biotech Co., Ltd. (Shanghai, China). The nucleotide sequences and chromatograms were viewed using the EditSeq 5.0 and Chromas 2.4 program, respectively. After aligning, consensus sequences were compared to similar sequences in the GenBank database using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/blast/) to determine the species, genotype, or assemblages of the protozoans.

2.4. Statistical analysis

Epidemiological data were organized in Microsoft Excel and analyzed using the R programming language. Chi-squared tests and Odds ratios (OR) with 95 % confidence intervals were calculated, and variables with p ≤ 0.05 in univariate analysis were further assessed using multivariable logistic regression. Associations were considered statistically significant at p ≤ 0.05.

3. Results

3.1. Molecular occurrence and characterization of Cryptosporidium spp.

The overall prevalence of Cryptosporidium spp. was 8.1 % (16/197) in cats and 4.2 % (5/120) in dogs. Molecular analysis identified 16 feline and 5 canine Cryptosporidium isolates. Among cats, C. felis was the most common species (81.3 %), followed by C. baileyi (12.5 %) and C. canis (6.3 %). In dogs, all positive isolates were C. canis (100 %). For C. felis, eight isolates were found in males and three in females, with six cats aged under one year and five aged 1–2 years. The single C. canis infection in a cat involved a male aged under one year. Two C. baileyi isolates were also found in males, one younger than one year and the other between one and two years old. Among dogs, C. canis was detected in three males and two females, with three dogs under one year of age and two aged 1–2 years.

3.2. Risk factors for Cryptosporidium spp.

In cats, a higher prevalence was observed in animals from Gazipur (12.1 %) than from Dhaka (7.3 %), and in males (10.6 %) than in females (5.4 %). Age-related variation was limited, with infections detected mainly in cats younger than two years and none in cats older than two years. However, these differences were not statistically significant in the univariate analysis (Table 1, all p > 0.05). Breed, deworming status, food type, and outdoor access were also not significantly associated. Contrarily, hunting behavior and cohabitation status were significantly associated with Cryptosporidium occurrence in cats. Cats without hunting behavior exhibited a higher prevalence (11.6 %) than hunting cats (0 %; χ² = 7.445, p = 0.006), and cats not cohabiting with other animals had a higher prevalence (10.5 %) compared with cohabiting cats (0 %; χ² = 5.16, p = 0.023). However, logistic regression analysis revealed that none of the assessed factors remained significantly correlated with Cryptosporidium occurrence in cats after multivariable adjustment. In dogs, Cryptosporidium prevalence was uniformly low across all examined variables, ranging from 0 % to 9.38 % (Table 2). Slightly higher prevalence was observed in dogs from Gazipur (4.4 %) than in Dhaka (4.1 %), in males (4.3 %) than females (4 %), and in dogs younger than one year (9.4 %) compared with older age groups; however, none of these differences were statistically significant in either univariate or multivariable analyses.

Table 1.

Prevalence of Cryptosporidium spp. and G. duodenalis in cats and their association with different variables assessed by the χ² test and multivariable logistic regression analysis.

a	Cryptosporidium spp.							G. duodenalis
	N	%	χ2	p	OR	95 % CI	p	%	χ2	p	OR	95 % CI	p
Location
Dhaka	164	7.3	0.850	0.357	Ref.			31.7	1.442	0.230	Ref.
Gazipur	33	12.1			1.04	0.25–3.63	0.956	21.2			1.60	0.62–4.55	0.350
Sex
Female	93	5.4	1.780	0.182	Ref.			24.7	2.286	0.131	Ref.
Male	104	10.6			1.61	0.50–5.67	0.435	34.6			1.44	0.75–2.80	0.279
Age
>2 years	17	0.0	1.747	0.417	Ref.			35.3	0.603	0.740	Ref.
<1 year	74	9.4			6.18 × 107	0.04–N/A	0.998	31.1			1.50	0.43–4.98	0.515
1–2 years	106	8.1			8.11 × 107	3.72 × 10−157–N/A	0.997	27.0			1.37	0.67–2.87	0.392
Breed
Cross	45	6.7	0.166	0.684	Ref.			33.3	0.318	0.573	Ref.
Local	152	8.6			1.34	0.31–7.61	0.714	28.9			1.61	0.67–3.85	0.278
Deworming status
No	121	9.9	1.355	0.244	Ref.			28.9	0.157	0.692	Ref.
Yes	76	5.3			1.26	0.30–4.60	0.735	31.6			1.03	0.47–2.24	0.934
Food type
Cat food	12	0.0	4.260	0.235	Ref.			0.0	9.148	0.027*	Ref.
Cooked meat	127	11.0			1.14 × 108	2.74 × 10−166–N/A	0.998	31.5			1.76 × 107	6.56 × 10−25–NA	0.988
Mixed	54	3.7			7.45 × 107	1.26 × 10−151–N/A	0.998	29.6			1.29 × 107	8.75 × 10−25–NA	0.988
Raw animal organs	4	0.0			5.58 × 107	2.64 × 10−179–3.56 × 10192	0.999	75.0			1.25 × 108	1.31 × 108–1.59 × 10185	0.986
Source
Stray	35	0.0	5.113	0.164	Ref.			34.3	0.618	0.892	Ref.
Breeder	27	7.4			5.75 × 107	2.59 × 10−102–N/A	0.996	25.9			2.30	0.60–9.34	0.232
Family or friends	92	8.7			3.23 × 107	1.05 × 10−99–N/A	0.996	30.4			1.90	0.60–6.71	0.293
Shelter	43	14.0			4.59 × 107	2.07 × 10−102–N/A	0.996	27.9			1.84	0.48–7.62	0.384
Outdoor access
Yes	11	0.0	1.030	0.310	Ref.			18.2	0.769	0.381	Ref.
No	186	8.6			3.36 × 107	3.90 × 10−144–N/A	0.998	30.6			3.83	0.72–32.80	0.151
Hunting status
Yes	59	0.0	7.445	0.006*	Ref.			33.9	0.626	0.429	Ref.
No	138	11.6			4.12 × 107	5.04 × 10−31–1.25 × 1046	0.996	28.3			1.04	0.34–3.05	0.938
Cohabitation
Yes	45	0.0	5.156	0.023*	Ref.			35.6	0.874	0.350	Ref.
No	152	10.5			7.89 × 106	7.81 × 10−71–NA	0.996	28.3			1.81	0.61–5.73	0.295

Extreme OR values (N/A, ≥10⁷) result from absent infections in one categorical group. *Statistically significant.

Table 2.

Prevalence of Cryptosporidium spp. and G. duodenalis in dogs and their association with different variables assessed by the χ² test and multivariable logistic regression analysis.

Variables	Cryptosporidium spp.							G. duodenalis
	N	%	χ2	p	OR	95 % CI	p	%	χ2	p	OR	95 % CI	p
Location
Dhaka	97	4.12	0.002	0.961	Ref.			23.71	0.448	0.503	Ref.
Gazipur	23	4.35			1.02	0.01–51.30	0.994	30.43			1.68	0.30–9.57	0.551
Sex
Female	50	4	0.006	0.938	Ref.			38	7.726	0.005*	3.72	1.45–10.08	0.008*
Male	70	4.29			1.06	0.13–12.06	0.958	15.71			Ref.
Age
>2 years	31	0	3.584	0.167	Ref.			29.03	0.415	0.813	1.33	0.43–4.03	0.609
<1 year	32	9.38			1.29 × 108	NA	0.995	25			1.12	0.35–3.45	0.839
1–2 years	57	3.51			3.07 × 107	NA	0.995	22.81			Ref.
Breed
Cross	64	4.69	0.093	0.760	Ref.			26.56	0.179	0.673	Ref.
Local	56	3.57			1.20	0.05–33.60	0.905	23.21			1.95	0.49–8.30	0.347
Deworming status
Yes	38	2.63	0.328	0.567	Ref.			28.95	0.462	0.497	Ref.
No	82	4.88			9.18	0.27–791.48	0.256	23.17			2.10	0.52–9.11	0.304
Food type
Raw animal organs	31	3.23	0.243	0.886	Ref.			19.35	0.901	0.637	Ref.
Cooked meat	73	4.11			1.81	0.09–49.11	0.679	26.03			1.68	0.44–6.57	0.444
Mixed	16	6.25			2.69	0.04–202.31		31.25			1.14	0.18–6.91	0.889
Water source
Filtered water	20	5	0.253	0.881	Ref.			35	2.091	0.352	Ref.
Tap	95	4.21			3.14 × 107	NA	0.999	22.11			1.13	0.06–47.51	0.939
WASA	5	0			6.58	NA		40			3.51	0.09–316.86	0.527
Outdoor access
No	21	4.04	0.023	0.880	Ref.			22.22	2.328	0.127	Ref.
Yes	99	4.76			3.36	0.06–280.85	0.541	38.1			2.14	0.47–9.77	0.317
Hunting status
No	6	0	0.275	0.600	Ref.			33.33	0.234	0.629	Ref.
Yes	114	4.39			3.15 × 1015	NA–NA	0.996	24.56			3.16	0.28–48.13	0.372
Cohabitation
No	102	3.92	0.102	0.749	Ref.			22.55	2.179	0.140	Ref.
Yes	18	5.56			1.90 × 108	NA–NA	0.997	38.89			6.74	0.43–287.49	0.228

Extreme OR values (N/A, ≥10⁷) result from absent infections in one categorical group. *Statistically significant.

3.3. Molecular occurrence and multilocus genotyping of G. duodenalis

PCR amplification and sequencing of the bg, gdh, and tpi genes revealed an overall prevalence of G. duodenalis of 29.9 % (59/197) in cats and 25 % (30/120) in dogs. At the gene-specific level, the tpi locus showed the highest detection rate of G. duodenalis in both cats (28.9 %) and dogs (24.2 %), while the gdh locus exhibited the lowest detection (6.6 % in cats and 8.3 % in dogs). Multilocus genotyping revealed the presence of assemblages A (80 %), F (13 %), and C (5 %) in cats, and A (53 %), C (17 %), and D (10 %) in dogs. Mixed infections were uncommon in cats, with only 2 % exhibiting a combination of A and F, but were more frequent in dogs (20 %), involving assemblage combinations such as A/C, A/D, and C/D. Most isolates were identical to reference sequences; however, some carried nucleotide substitutions at various positions, indicating minor intra-assemblage genetic variation.

3.4. Risk factors for G. duodenalis

In cats, prevalence was higher in Dhaka (31.7 %) than in Gazipur (21.2 %), whereas in dogs, it was higher in Gazipur (30.4 %) than in Dhaka (23.7 %), though these differences were not statistically significant. Male cats exhibited a higher prevalence (34.6 %) compared to females (24.7 %). In dogs, females showed a significantly higher prevalence (38 %) than males (15.7 %). Age-related trends in cats showed slightly higher prevalence in animals older than one year (35.3 %) than in younger cats (31.1 %). However, no clear age-related pattern was observed in dogs (25 % in <1 year, 22.8 % in 1–2 years, and 29 % in >2 years). Among management-related factors, diet type was significantly associated with G. duodenalis infection in cats (p = 0.027), with no infections detected in cats fed commercial food, while higher prevalence was observed in cats fed cooked meat (31.5 %), mixed diets (29.6 %), or raw organs (75 %) (Table 1). In dogs, sex was the only significant factor (p = 0.005), with females showing a higher likelihood of infection (OR = 3.7, 95 % CI: 1.45–10.08). No significant associations were observed for other variables in either species (Table 1, Table 2). However, multivariable logistic regression confirmed that sex was a significant predictor of G. duodenalis infection in dogs (Table 2).

4. Discussion

The prevalence of Cryptosporidium spp. in cats (8.1 %) and dogs (4.2 %) observed in this study is broadly consistent with reports from many regions in Asia and Europe, where infection rates in companion animals typically remain below 10 %. Similar prevalence levels have been reported from East Asia and parts of Europe (Ayan et al., 2024, Jian et al., 2025), whereas substantially higher rates have been documented in some regions of the Middle East and Latin America, likely reflecting differences in environmental contamination, animal management, and diagnostic sensitivity rather than true geographic variation (Tangtrongsup et al., 2020; Procesi et al., 2022). In contrast, G. duodenalis was detected at comparatively higher frequencies in both cats (29.9 %) and dogs (25 %), exceeding pooled global prevalence estimates for cats and dogs (Bouzid et al., 2015). Increased G. duodenalis prevalence has been reported across multiple regions, including Asia, Europe, and Africa, particularly in urban or high-density settings, suggesting fecal-oral transmission and persistent environmental contamination with cysts (Bouzid et al., 2015; Ceylan et al., 2024). Variations in the reported prevalence of both parasites across studies are likely influenced by geographical differences, host age and health status, diagnostic sensitivity, and environmental or management practices (Salant et al., 2020).

In this study, female dogs showed a significantly higher prevalence of G. duodenalis than males, consistent with studies from China, Korea, and Germany, where female sex was considered a risk factor likely linked to physiological and hormonal changes during pregnancy and the postpartum period (Li et al., 2019). However, several studies have reported no significant sex-related differences in G. duodenalis, reflecting population and environmental variability (Idan and Al-Hasnawy, 2023). In the case of Cryptosporidium, sex was not significantly associated in cats and dogs from this study, consistent with reports from Iran and Jordan (Karimi et al., 2023; Mukbel et al., 2025). However, Lider et al. (2025) identified sex as a significant risk factor, with higher cryptosporidiosis rates in female cats.

Geographic location was not associated with the prevalence of Cryptosporidium or G. duodenalis in cats and dogs, consistent with reports from diverse regions indicating limited spatial effects on infection risk (Karimi et al., 2023). Instead, infection patterns appear to be driven primarily by environmental contamination and host-related factors rather than geography (de Oliveira et al., 2021). Likewise, age, breed, deworming status, water source, outdoor access, hunting behavior, and cohabitation were not significant predictors of infection, although contrasting associations have been reported elsewhere, likely reflecting differences in environmental exposure and management practices (Tangtrongsup et al., 2020; Taghipour et al., 2021). Overall, these findings underscore the dominant role of ecological and environmental factors over individual host traits in shaping the epidemiology of Cryptosporidium and G. duodenalis in cats and dogs.

Molecular analysis revealed species-level diversity among Cryptosporidium isolates. C. felis was the predominant species in cats, aligning with global reports (de Oliveira et al., 2021; Karimi et al., 2023). Interestingly, C. felis has also been reported in captive fishing cats in Bangladesh (Karim et al., 2021), suggesting its widespread distribution among both domestic and wild felids in the country. The detection of C. baileyi in two cat samples, an avian-adapted species, suggests possible environmental contamination or transient infection (Helmy and Hafez, 2022). While C. canis is typically dog-specific, its occasional occurrence in cats likely results from environmental exposure or close contact with dogs (Alali and Alkhaled, 2023). All dog isolates were identified as C. canis, consistent with host preference (Jian et al., 2014). The reporting of these Cryptosporidium species raises concerns about potential zoonotic transmission, as they have also been identified in humans (Hao et al., 2024).

G. duodenalis assemblage A was the predominant genotype in cats, consistent with reports from Italy and Mexico (Procesi et al., 2022; Veyna-Salazar et al., 2023). The dominance of the zoonotic assemblage A suggests that companion animals may serve as potential reservoirs for human giardiasis, as this assemblage often causes human giardiasis in both developing and developed countries (Feng and Xiao, 2011). It has previously been reported in 34 % of children (Li et al., 2023) and 15.8 % of hospitalized diarrheal patients in Bangladesh (Haque et al., 2005). This assemblage has also been identified in 11.8 % of calves (Li et al., 2023), as well as in captive waterbucks, spotted deer, Nilgiri langurs, and giraffes in Bangladesh (Karim et al., 2021). The detection of feline assemblage F and canine assemblage C in cats indicates the coexistence of host-specific genotypes alongside cross-host or potentially zoonotic genotypes. Similar cross-host occurrences of assemblages F and C in cats have been reported in China, suggesting possible interspecies transmission (Li et al., 2024). In dogs, assemblage A predominated, consistent with Sun et al. (2023), although assemblages C and D, typically more common globally, were also detected in this study. Mixed infections, primarily involving assemblages A, C, and D, were common in dogs, reflecting a higher exposure risk due to cohabitation, crowding, or the simultaneous presence of multiple assemblages (Sui et al., 2022).

These findings represent critical surveillance gaps in the public health infrastructure of Bangladesh. Previous systems used to rely on microscopic methods that miss species- and assemblage-level diversity (Mahmud et al., 2014; Nipa et al., 2022), while high prevalence in asymptomatic cats and dogs suggests unrecognized human infection sources in densely populated urban settings. The molecular approach revealed substantial genetic diversity and zoonotic potential that are often missed by routine diagnostics. These findings highlight the need for integrated surveillance across veterinary and human health sectors. Targeted interventions are therefore warranted, including pet owners' education on raw meat feeding and pet hygiene, molecular screening of symptomatic animals with referral pathways to human health services, strengthened regulation of pet ownership and waste management, and integration of Cryptosporidium and G. duodenalis surveillance into national infectious disease systems. A pragmatic One Health approach incorporating molecular epidemiology and risk factor assessment is essential to identify transmission hotspots and guide interventions in high-burden urban areas and vulnerable populations.

A notable limitation of this study is the lack of systematic documentation of clinical signs and gastrointestinal health status in sampled animals. Since Cryptosporidium and G. duodenalis infections are frequently subclinical in pets (Taghipour et al., 2020), enabling oocyst/cyst shedding without apparent disease, future studies should document clinical manifestations to assess infection-disease associations and stratify transmission risk based on symptom status.

This first molecular epidemiological assessment in Bangladesh establishes a crucial baseline demonstrating that cats and dogs are important reservoirs for human-pathogenic Cryptosporidium and G. duodenalis, with zoonotic species and assemblages circulating at substantial prevalence. The identification of modifiable risk factors, particularly pet food, provides actionable targets for public health intervention and compelling evidence for implementing integrated, One Health-informed strategies to reduce zoonotic disease burden.

Availability of data and materials

All unique gene sequences of Cryptosporidium spp. and G. duodenalis from this study have been archived in the GenBank database of the National Center for Biotechnology Information (NCBI) under unique accession numbers. Accession numbers for the SSU rRNA gene of Cryptosporidium spp. are PX101837- PX101846 for cats and PV948062 for dogs. For G. duodenalis in cats, the accession numbers range from PX118515 to PX118521 for the bg gene, PX118522 to PX118524 for the gdh gene, and PX118525 to PX118529 for the tpi gene. In dogs, the accession numbers range from PV983403 to PV983408 for the bg gene, PV983409 to PV983413 for the gdh gene, and PV983414 to PV983424 for the tpi gene.

CRediT authorship contribution statement

Ainun Nahar: Writing – original draft, Investigation, Formal analysis, Data curation. Md. Farhan Hasan: Writing – original draft, Visualization, Software, Investigation, Formal analysis, Data curation. Anas Bin Harun: Writing – review & editing, Visualization, Software, Investigation, Formal analysis, Data curation. Abdullah Al Bayazid: Writing – review & editing, Resources, Investigation, Data curation. Tania Sultana: Resources, Investigation, Writing – review & editing. Jinnat Rehena: Investigation, Data curation, Writing – review & editing. Joynti Saha: Investigation, Data curation, Writing – review & editing. S.H.M. Faruk Siddiki: Writing – review & editing, Supervision, Formal analysis. Md. Mizanur Rahman: Writing – review & editing, Supervision, Project administration. 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, Project administration.

Ethics statement

The Animal Research Ethics Committee (AREC) of Gazipur Agricultural University (GAU), Bangladesh, reviewed and approved the methodology of this study (reference: FVMAS/AREC/2023/36). After clearly explaining the research objectives to the pet owners, written informed consents were obtained before data and sample collection.

Funding

This research was partially funded by the Ministry of Science and Technology (MoST), Bangladesh (Grant No. SRG-241011 and SRG-231003). 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.