Introduction

Cryptosporidium and Giardia are globally significant protozoan parasites frequently associated with severe foodborne and waterborne outbreaks1,2. These pathogens contribute substantially to morbidity and mortality worldwide, particularly in low-resource settings where they disproportionately impact vulnerable populations, including children, immunocompromised individuals, and newborn animals3,4,5,6. Both parasites exhibit a wide range of vertebrate hosts, infecting humans, livestock, wildlife, and birds, causing self-limited diarrhea alongside other clinical manifestations7,8,9,10,11. Their zoonotic potential and diverse transmission pathways, including zoonotic, foodborne, and waterborne routes, underscore their relevance as a critical One Health concern, highlighting the interconnectedness of human, animal, and environmental health12,13,14.

Cryptosporidium oocysts and Giardia cysts are highly resilient in the environment, persisting in diverse matrices such as soil, water, and food. Their transmission is facilitated through contaminated tap water, bottled water, surface water, ground water, and irrigation systems, posing significant public health and environmental challenges15,16,17,18. Farm animals, particularly cattle, play a pivotal role in the epidemiology of these protozoa, as young calves serve as major reservoirs, shedding millions of infectious oocysts and cysts into the environment, contaminating water sources, and amplifying the zoonotic transmission risks8,19,20,21. The remarkably low infective dose of Giardia, fewer than 10 cysts, further exacerbates the ease of transmission, complicating public health and environmental management efforts20,21,22. The infective dose of Cryptosporidium parvum ranges from 5.8 to 16.6 oocysts23 yet a single infected host can shed over 3 × 1010 oocysts into the environment24 creating a significant potential reservoir for environmental contamination25. To date, more than 40 Cryptosporidium spp. have been identified in mammals, reptiles, birds, fish, and amphibians26. Of the approximately 20 species known to infect humans, C. parvum and Cryptosporidium hominis are the most prevalent, accounting for over 90% of human cases globally3. In cattle, the predominant species include C. parvum, Cryptosporidium bovis, Cryptosporidium andersoni, and Cryptosporidium ryanae26. Among these, C. parvum is particularly notable for its broad host range and significant zoonotic potential. Giardia intestinalis (synonyms: Giardia lamblia and Giardia duodenalis) is a species complex comprising eight genetically distinct assemblages (A–H). Assemblages A and B are of particular concern due to their low host specificity, allowing them to infect both humans and a wide variety of animal species22,27,28. Co-infections involving Cryptosporidium and Giardia are increasingly documented in both humans and animals, particularly in regions with inadequate sanitation or environmental contamination20,29,30. Emerging evidence suggests that co-infections may exacerbate clinical severity, leading to prolonged diarrhea, persistent inflammation, and malnutrition, especially in newborn animals31,32. The concurrent presence of both pathogens within a host may compromise immune responses, potentially facilitating the colonization or persistence of one pathogen by the other, thereby complicating disease management and treatment outcomes30. Furthermore, their simultaneous detection in environmental matrices, such as irrigation water and soil, underscores the heightened risk of widespread contamination and transmission10,15.

Cryptosporidiosis is endemic in cattle worldwide, with reported prevalence rates ranging from 11.7 to 78%, particularly affecting pre-weaned calves33,34,35,36. In humans, the global prevalence of cryptosporidiosis has been estimated at 14.1% in high-income countries and up to 31.5% in low-income countries37,38. Human infections with Giardia spp. are similarly widespread, ranging from 2 to 5% in developed nations to 20–30% in developing regions39,40,41,42. In cattle, the pooled prevalence of giardiasis has been estimated at 24% based on microscopic examination43. Both Cryptosporidium and Giardia are well-recognized as major pathogens responsible for numerous foodborne and waterborne outbreaks1,44,45,46. Globally, the proportion of waterborne outbreaks attributed to Cryptosporidium increased markedly to 77.4% between 2017 and 2022, while those caused by Giardia declined significantly to 17.1% during the same period47.

Cryptosporidiosis and giardiasis pose significant One Health challenges due to their intricate interplay across human, animal, and environmental systems13,14. Accurate diagnosis requires a combination of microscopical and molecular techniques to identify species and genotypes, which are critical for understanding host specificity, pathogenicity, and zoonotic potential30,48,49. Elucidating the prevalence, risk factors, and potential synergistic effects of co-infection is essential for designing effective surveillance, prevention, and control strategies within the One Health framework7,32,50.

This study aimed to comprehensively assess the prevalence of cryptosporidiosis, giardiasis, and their co-infections in Beni-Suef Governorate, Egypt, using microscopical examination followed by genetic identification of detected species. Additionally, it seeks to identify associated risk factors in cattle fecal samples, emphasizing their role in the epidemiology of these protozoan infections.

Materials and methods

Study area

The study was conducted in Beni-Suef province (29.0667° N, 31.0833° E) in northern Upper Egypt (Fig. 1), an agricultural hub where cattle rearing plays a key economic role. Cattle are raised for milk and meat, providing vital income and resources for local households. The region’s semi-arid climate, with hot summers and mild winters, shapes cattle farming practices, with farmers using irrigated pastures and crop residues for feed. The nearby Nile River ensures reliable water access, supporting cattle health and productivity.

Fig. 1
figure 1

A map of Egypt showing the provinces, with Beni-Suef province highlighted in orange to indicate where the samples were collected.

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Sampling

A cross-sectional study was conducted in 2023 and 2024, involving 970 cattle. Fecal samples were randomly collected from cattle owned by smallholders, private farms, and at the Beni-Suef slaughterhouse. Various risk factors were recorded for each animal, including sex (female and male), age categories (< 2 months, 2–4 months, 4–6 months, and > 6 months), seasons (autumn, spring, summer, and winter), and observed fecal consistency. Rectal samples were collected from each animal and placed into clean, labeled plastic containers. The samples were then transported in an icebox to the Parasitology and Immunology Laboratory, Department of Parasitology and Animal Diseases, NRC, Egypt, on the collection day49. In addition, 24 irrigation water samples (20 L each) were collected from the same locations using sterile polypropylene containers. These water samples were transported to the Environmental Parasitology Laboratory, Water Pollution Research Department, NRC, Giza, Egypt, on the same day51.

Parasitological examination

Fecal examination

Macroscopical examination

Fecal samples were examined macroscopically to assess consistency, color abnormalities, and the presence of blood, mucus, or other unusual components, following the methodology described by Zajac et al.52.

Microscopical examination

The fecal samples were filtered through two layers of gauze to remove large particles. Approximately 2 mg of feces was then mixed with a drop of normal saline (0.85% NaCl) and a drop of Lugol’s iodine solution, and the mixture was spread on a clean glass slide. Each specimen was examined under a light microscope (LEICA Imaging Systems Ltd., England) at 100× and 400× magnification for the morphological identification of Giardia cysts and trophozoites42,49. Cryptosporidium spp. were identified using the Modified Ziehl Neelsen (MZN) staining technique, as described by Henriksen and Pohlenz53. MZN-stained slides were examined at 400× and 1000× magnification54. The severity of infection was assessed by counting Cryptosporidium oocysts per field at 1000× magnification, following the criteria outlined by Anderson and Bulgin55: mild (1–5 oocysts/field), moderate (6–20 oocysts/field), and severe (more than 20 oocysts/field). Samples were stored at 4 °C in an equal volume of 2.5% potassium dichromate solution (Sigma-Aldrich, Canada) until molecular identification56.

Water examination

Each water sample was filtered using a stainless-steel pressure filter holder (Sartorius, Germany) fitted with a nitrocellulose membrane 142 mm diameter, 0.45 μm pore57. The membrane filters were washed three times with sterile saline, and the washing solution was centrifuged at 2000 rpm for 5 min58. The supernatants were discarded, and the resulting sediments were separately collected in sterile Eppendorf tubes. Parasitological examination was performed as previously described, and the samples were subsequently stored at −20 °C for molecular identification.

Molecular screening

DNA extraction

DNA was extracted from heavily infected fecal and water samples that tested positive during microscopic examinations; 200 µL of each fecal sample and 500 µL of each water sample, containing concentrated oocysts, using the QIAamp® DNA Stool Mini Kit (Qiagen GmbH, Hilden, Germany), following the manufacturer’s protocol. Before extraction, the samples underwent five freeze-thaw cycles, alternating between liquid nitrogen and a 95 °C water bath. The DNA concentration of each sample was measured using a Q9000 microvolume spectrophotometer (Quawell, USA). The extracted DNA was stored at − 20 °C until further analysis for pathogen screening.

Screening of pathogens DNA by standard PCR

All extracted DNA samples were screened using PCR with universal primers targeting the Cryptosporidium spp. 18S rRNA59 and Giardia spp. β-giardin (bg) gene60. The PCR assays were conducted using Emerald Amp GT mastermix™ (Takara) in a BIO-RAD Thermal Cycler (BIO-RAD, Singapore). Amplification conditions for both Cryptosporidium and Giardia followed the protocols outlined by Yusof et al.59, Cacciò et al.60 (Table 1). Each PCR run included positive controls (genomic Cryptosporidium and Giardia DNA) and negative controls (molecular-grade water). Amplification products were verified by electrophoresis on a 1% agarose gel stained with Red Safe and visualized using a UV transilluminator. A 100 bp DNA ladder (Fermentas, Thermo Fisher Scientific) was used to determine the size of the PCR products.

Table 1 Oligonucleotide sequences of primers used for PCR and sequencing.
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Table 2 Epidemiological risk factors associated with the prevalence of cryptosporidiosis and giardiasis.
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Sequencing and phylogenetic analyses

PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Germany) following the manufacturer’s instructions. Sequencing of the purified products was carried out on an ABI 3130 automated sequencer (Applied Biosystems, USA) using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The resulting sequences were assembled and refined using the Chromas Pro program (ChromasPro 1.7, Technelysium Pty Ltd., Tewantin, Australia). After submission to GenBank, the corrected sequences for Cryptosporidium spp., and Giardia spp. were compared to existing sequences in the GenBank database using NCBI BLASTn (http://blasdt.ncbi.nlm.nih.gov/Blast.cgi). The consensus sequences were aligned with reference sequences from GenBank using CLUSTAL W v1.8361. Phylogenetic trees were constructed using the Maximum Likelihood method in MEGA X, based on the Tamura-Nei model, with 1,000 bootstrap replicates to ensure statistical reliability62,63.

Data analysis

The impact of various risk factors, including sex, age, season, and fecal consistency, on the prevalence of Cryptosporidium, Giardia, and co-infections were assessed using the chi-square (χ2) test in SAS software, Version 9.4 (SAS Institute Inc., Cary, NC, USA). Statistical significance was determined at a threshold of P < 0.05.

Results

Prevalence of cryptosporidiosis, giardiasis, and their Co-infection

Parasitological examination revealed that out of 970 cattle examined, 654 animals (67.42%) tested positive for one or more parasitic infections. Cryptosporidium mono-infections had the highest prevalence, affecting 414/970 cattle (42.68%), followed by co-infections of Cryptosporidium with Giardia sp. in 124/970 cases (12.78%) and Giardia mono-infections in 116/970 cases (11.96%; Table 2). In irrigation water samples, Cryptosporidium sp. was more prevalent, detected in 2 out of 24 samples (8.33%), whereas Giardia sp. was identified in 1 out of 24 samples (4.16%).

Epidemiological risk factors associated with cryptosporidiosis, giardiasis, and their Co-infection

Epidemiological analysis demonstrated that the prevalence of cryptosporidiosis was significantly associated with sex, age, seasonal variation, and fecal consistency. The highest occurrence was observed among females (45.21%; P = 0.0180), calves less than 2 months of age (47.68%; P < 0.0001), during autumn (51.28%; P < 0.0001), and in diarrheic cases (46.88%; P < 0.0001). In contrast, the prevalence of giardiasis was significantly influenced by age and seasonal variation, with the highest rates recorded in calves aged between 4 and 6 months (15.38%; P < 0.001) and during spring (19.09%; P = 0.0033). However, no statistically significant differences were found in giardiasis prevalence concerning sex or fecal consistency (Table 2). For cases of co-infection involving both pathogens, age, seasonal variation, and fecal consistency also emerged as significant determinants. The highest co-infection rates were detected in calves aged between 4 and 6 months (15.38%; P < 0.0001), during spring (25.45%; P < 0.0001), and among diarrheic cases (15.07%; P < 0.001). Similar to giardiasis, co-infection prevalence was not significantly associated with sex (Table 2).

Overall, the infection rates for both pathogens combined were significantly linked to all examined risk factors. Specifically, the highest prevalence was noted among females (68.19%; P = 0.0380), calves less than 2 months of age (70.66%; P = 0.0160), during spring (82.27%; P < 0.0001), and in diarrheic cases (72.98%; P < 0.0001).

Molecular and phylogenetic analyses of cryptosporidiosis and giardiasis

All microscopically positive samples were screened by species-specific primers, the 18S rRNA gene for Cryptosporidium spp., and the β-giardin primer for Giardia (S1 and S2 Appendix). The 18S rRNA gene sequencing confirmed the presence of four Cryptosporidium species: Cryptosporidium hominis and Cryptosporidium bovis in cattle feces, and Cryptosporidium ubiquitum and Cryptosporidium ryanae in irrigation water. BLAST analysis revealed a single genotype for each species, consistent across different seasons. C. hominis (GenBank: PQ149132.1) showed 100% identity (464/464 bp) with C. hominis detected in the feces of rhesus macaques (Macaca mulatta), an Old World monkey species, in Bangladesh (GenBank: MK982514). Similarly, C. bovis (GenBank: PQ149134.1) demonstrated 100% identity (453/453 bp) with C. bovis detected in the feces of dairy cattle in China (GenBank: MF074601). In irrigation water, C. ubiquitum (GenBank: PQ149133.1) exhibited 100% similarity (462/462 bp) to C. ubiquitum identified in goat feces from China (GenBank: MN833283), while C. ryanae (GenBank: PQ149135.1) showed 100% similarity (453/453 bp) to C. ryanae identified in calf feces from Ethiopia (GenBank: KT922233). Phylogenetic analysis of Cryptosporidium spp. indicated that our sequences clustered within the same clade as reference species (Fig. 2).

Fig. 2
figure 2

18S rRNA-based phylogenetic tree of Cryptosporidium spp. The Maximum Likelihood method was constructed based on the Tamura-Nei model with 1000 bootstrap replicates.

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All Giardia-positive samples were identified as G. intestinalis using β-giardin primers. BLAST analysis showed one genotype of G. intestinalis (GenBank: PP316111.1), with 100% identity (474/474 bp) to G. intestinalis detected in human feces from Brazil (GenBank: KX015671). However, the irrigation water sample yielded low-quality sequences, making identification challenging. Based on the β-giardin gene, the phylogenetic tree showed that our sequence clustered with other G. intestinalis of the assemblage A clade (Fig. 3).

Fig. 3
figure 3

β-giardin -based phylogenetic tree of Giardia spp. The Maximum Likelihood method was constructed based on the Tamura-Nei model with 1000 bootstrap replicates.

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Discussion

Understanding the distribution and risk factors of Cryptosporidium and Giardia is crucial due to their significant role in zoonotic transmission and environmental contamination. Cattle, particularly pre-weaned calves, are major reservoirs, shedding large quantities of (oo)cysts, which contribute to environmental contamination and pose serious public health and veterinary risks22,64. Accurate species identification at the herd level is essential for implementing effective treatment and prevention strategies65. Therefore, this study aimed to investigate the prevalence of Cryptosporidium and Giardia infections, as well as co-infections, in cattle feces and nearby irrigation water in Beni-Suef Governorate, Egypt, using an integrated diagnostic approach combining microscopy and molecular techniques.

In this study, microscopic analysis revealed an overall infection rate of 67.42% for bovine cryptosporidiosis and giardiasis, with Cryptosporidium oocysts detection in 42.68%, Giardia cysts in 11.96%, and co-infections in 12.78% of the examined cattle. The prevalence of cryptosporidiosis was comparable to the 46% infection rate reported in cattle from Cairo, Giza, and Beni-Suef66 but was relatively higher than rates recorded in other Egyptian Governorates, such as 34.33% in Minufiya6738.27% in Upper Egypt49 and 24.67% in Kafr ElSheikh, 14.29% in Qalyubia, and 17.14% in Gharbia68. Globally, cryptosporidiosis prevalence varies widely, with reported rates of 1.61% in China69 4.4% in Korea70, 8.3% in Kenya71 10% in Ethiopia72 13.7% in Algeria73 19.23% in Saudi Arabia48 27.3% in Canda74 and 55.4% in Austria75. These variations may be influenced by hygienic practices, infection severity, geographic location, cattle breed, animal age, seasonal factors, and sample size76.

Similarly, the giardiasis infection rate (11.96%) observed in this study is consistent with a previous report from El-Dakahlia, El-Gharbia, and Damietta Governorates, where a prevalence of 13.3% was recorded77. Globally, giardiasis prevalence in calves varies considerably, with reported rates of 2.1% and 2.2% in China69,78 5.6% and 12.7% in Korea70,79 5.7% in Bangladesh80 7.5% in Brazil81 27.1% in Austria75 27.5% in Algeria73 33.5% in the USA82 39% in Ethiopia72 and 42% in Canada74. These variations in prevalence may be attributed to differences in geographic location, climate, herd management practices, diagnostic methods, and sample size27.

Co-infection with Cryptosporidium and Giardia was detected in 12.78% of studied cattle, consistent with previous reports indicating that such co-infections are common in bovines83. A higher prevalence (36%) was recorded in Ismailia Governorate, Egypt84 while lower rates were reported in Canada 8.5%74 and Austria 11.8%75. Moreover, several studies suggested a positive association between Cryptosporidium and Giardia infections, which may be linked to water contamination as a shared transmission route85,86,87.

Water was identified as a key risk factor for animal exposure to Cryptosporidium and Giardia in this study. Cryptosporidium spp. was detected in 2 of 24 irrigation water samples (8.33%), while Giardia was identified in 1 sample (4.16%). In Egypt, previous studies have reported Cryptosporidium and Giardia prevalence in water ranging from 5.2 to 80% and 13.6–100%, respectively83,86,87,88,89,90,91 with the highest prevalence observed in raw wastewater and the lowest in treated water83,90. Detecting these protozoan parasites in water remains challenging due to the complexity of the water matrix, the typically low concentration of (oo)cysts92 variations in contamination levels, and differences in water sources used for irrigation93.

The analysis of epidemiological risk factors revealed a significant association between animal sex and the prevalence of both overall infection and cryptosporidiosis, with female calves exhibiting higher infection rates. These findings are consistent with previous studies that have also reported a greater prevalence of cryptosporidiosis among female animals94,95,96. This increased susceptibility may be attributed to physiological and hormonal differences, as well as management practices such as the preferential retention of female calves for breeding, which may result in prolonged housing, higher stocking densities, and increased pathogen exposure.

In addition to sex, age also plays a critical role in infection susceptibility. Calves younger than 2 months showed higher morbidity rates for both overall infection and cryptosporidiosis, a trend that aligns with previous studies reporting increased susceptibility in this age group77,94,95,97. This heightened vulnerability is likely due to an underdeveloped and immature immune system96. Conversely, calves between 4 and 6 months of age exhibited a higher prevalence of giardiasis, which is consistent with findings indicating that giardiasis is most common in calves aged 2 months and older75,77,98.

Beyond age and sex, seasonal variations also influenced infection patterns. While both overall infection and giardiasis were more prevalent in the spring, cryptosporidiosis cases peaked in the autumn. These findings are somewhat variable across studies, as some have reported a higher prevalence of infection during the rainy season99 whereas others have noted increased cases in the summer100. These divergences may be attributed to regional differences in farming practices, environmental conditions, and the availability of resources to minimize contamination30,96.

Furthermore, clinical signs such as diarrhea were strongly associated with cryptosporidiosis, with diarrheic cattle exhibiting a higher prevalence of infection compared to non-diarrheic ones. This observation aligns with previous studies that have established diarrhea as a predominant clinical sign of cryptosporidiosis94,99,101,102.

Molecular analysis confirmed the presence of Cryptosporidium and Giardia species in cattle feces and irrigation water, underscoring the complex transmission dynamics of these protozoan parasites within the One Health framework22,103,104. Four Cryptosporidium species were identified, C. hominis and C. bovis in cattle feces, and C. ubiquitum and C. ryanae in irrigation water, highlighting the diversity of species circulating in animal and environmental reservoirs. Notably, this study represents the first detection of C. hominis in cattle in Egypt, a significant finding given that C. hominis is primarily associated with human infections105. Its presence in cattle suggests potential anthroponotic transmission, likely resulting from environmental contamination or direct human-cattle interactions, consistent with previous reports of cross-species transmission20. In Egypt, C. hominis has been documented in humans106,107 and recently in sheep66. Globally, C. hominis has been detected in various animal hosts, including cattle, sheep, goats, horses, donkeys, and camels105. The detection of C. bovis in cattle feces from Beni-Suef Governorate further supports the role of livestock as reservoirs, contributing to environmental contamination and potential zoonotic transmission. Previous studies in Egypt have reported C. bovis in cattle from different Governorates, including Ismailia108 Kafr El Sheikh33,109 Beheira, Menofia, Qaliubiya, Assiut, and Sohag110. Consistent with global trends, C. bovis is commonly detected in cattle populations, often with low or no occurrence of C. parvum, as observed in Sweden111 China 112 Australia113 and Canada114.

The identification of C. ubiquitum and C. ryanae in irrigation water underscores the significance of waterborne transmission pathways. To the best of our knowledge, this study represents the first molecular detection of both species in irrigation water in Egypt. Previously, only C. parvum and C. hominis have been reported in drinking water in the country86,90,115. Cryptosporidium ubiquitum is recognized as the most prevalent Cryptosporidium spp. in sheep and goats20,116,117,118 and is also emerging as a human pathogen119. In Egypt, C. ubiquitum has previously been detected in sheep120. Similarly, C. ryanae is primarily associated with cattle20,121 and has been reported in cattle33,77,108,109 and buffaloes108,109,122 in Egypt. The detection of these species in irrigation water suggests that runoff from livestock operations may be a significant source of environmental contamination. This finding highlights the urgent need for water quality monitoring and improved agricultural waste management strategies to mitigate the risk of protozoan transmission through irrigation systems.

In this study, the identification of assemblage A, further supports the risk of cross-species transmission. In Egypt, Giardia assemblage A has been previously reported in cattle77 humans42,123,124,125 and tape water in the Beni-Suef Governorate86. The inability to obtain high-quality sequences from irrigation water suggests that environmental factors, such as microbial competition or DNA degradation, may influence Giardia detectability in water sources93. Nevertheless, the presence of G. intestinalis in cattle feces, along with its previous detection in tap water from the same region86 indicates that livestock may serve as reservoirs, with potential transmission occurring through direct contact, fecal contamination of water sources, or consumption of contaminated agricultural products.

Conclusion

This study highlights the high prevalence of Cryptosporidium and Giardia infections in cattle and irrigation water in Beni-Suef Governorate, Egypt, underscoring the role of cattle as reservoirs and the risk of environmental contamination. The first detection of C. hominis in cattle and C. ubiquitum and C. ryanae in irrigation water in Egypt suggests potential anthroponotic and waterborne transmission pathways while confirming the presence of C. bovis and Giardia assemblage A in cattle. Risk factor analysis showed higher infection rates in females, young calves, and during spring, with diarrheic feces strongly linked to parasite shedding. These findings emphasize the need for enhanced surveillance, improved livestock management, and stricter water quality monitoring. This study has some limitations, including its cross-sectional design, limited PCR sensitivity due to low (oo)cyst counts, and the lack of direct evidence linking water contamination to animal infection. Further research is needed to clarify transmission pathways and assess long-term impacts. Implementing One Health strategies with targeted interventions is essential to reducing infection risks and environmental contamination.