Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. are common zoonotic protists able to cause diarrhea and other gastrointestinal disorders in a wide range of animal species including humans (1–3). Human infection outcomes vary largely from asymptomatic to severe manifestations and even death. The most frequent clinical signs are abdominal discomfort, anorexia, acute and chronic diarrhea, nausea, and weight loss. Fever, vomiting, and bloody stool are less common (4–6). Extraintestinal manifestations including urticaria and other allergic diseases have also been reported for some of them (7). All four pathogens are fecal-orally transmitted after accidental ingestion of their transmissive stages (cysts, oocysts, spores) directly through contact with infected humans or animals or indirectly via consumption of contaminated water or fresh produce (8, 9).

Cryptosporidium spp., G. duodenalis, Blastocystis sp., and E. bieneusi display a large intra-species genetic diversity with marked differences in host specificity, range, zoonotic potential and even pathogenicity (10–12). Dogs and cats are commonly infected with Cryptosporidium spp. and G. duodenalis (13, 14), being primarily infected by host-adapted species/genetic variants including Cryptosporidium canis and Cryptosporidium felis and G. duodenalis assemblages C, D, and F. Despite the risk of zoonotic transmission of Cryptosporidium spp. and G. duodenalis from domestic dogs and cats is typically regarded as low (15–17), the sporadic but constant reporting of human infections caused by canine- and feline-adapted species/genotypes of these pathogens suggest that the role of dogs and cats as sources of human cryptosporidiosis and giardiasis should not be overlooked (18, 19).

The stramenopile Blastocystis sp. is a highly polymorphic protozoal parasite of uncertain pathogenicity commonly detected in fecal samples of humans and several other animal species. The parasite encompasses at least 36 subtypes (ST; ST1-ST17, ST21, ST23-ST40) (11, 20, 21). Blastocystis sp. is typically reported at relatively low (7%–9%) carriage rates in dogs and cats globally (22). Both host species have been shown to carry zoonotic STs including ST1–8, ST10, and ST14 (22), although the occurrence of zoonotic transmission events seems rare (23). Furthermore, E. bieneusi is an obligate intracellular fungus-like parasite with high genetic diversity among mammalian and avian hosts (24). Nearly 600 genotypes have been described within E. bieneusi (12), of which zoonotic genotypes A, BEB6, D, and TypeIV have been found circulating in domestic dogs and cats (25).

Domestic dogs and cats can carry a large variety of bacterial, viral, and parasitic (including protist) pathogens which can be transmitted to humans through bites, scratches, saliva, urine, feces, or contaminated surfaces. Therefore, understanding the frequency and molecular diversity of these pathogens is important to assess their zoonotic potential and public health relevance. In Egypt, information on the epidemiology of intestinal protist species of public and veterinary health relevance in canine and feline populations is scarce. Most of the studies conducted to date were based on conventional microscopy as screening method, and only few assessed the frequency and diversity of species/genotypes at the molecular level (Table 1) (26–39). It is therefore essential to conduct periodical surveys to provide updated information on the current status of these pathogens in domestic animals, which might be helpful to reduce the risk of potential zoonotic transmission events to humans. Under this approach, this molecular study investigated the occurrence, genetic diversity, and zoonotic potential of Cryptosporidium spp., G. duodenalis, Blastocystis sp., and E. bieneusi infection in domestic dogs and cats in three geographical areas of Egypt.

Domestic dogs and cats can be a source of human infection by a wide diversity of viral, bacterial, parasitic, and fungal pathogens (52, 53). Those with unrestricted access to the outdoors might be at higher risk of pathogen exposure and represent overlooked reservoirs of zoonotic agents (54). Therefore, elucidation of the epidemiology and public health importance in these pathogens requires the use of genotyping and subtyping tools (14). Under these premises, this molecular-based study evaluated the occurrence and molecular diversity of four of the most common diarrhea-causing enteric protist parasites (Cryptosporidium spp., G. duodenalis, E. bieneusi, and Blastocystis sp.) in canine and feline populations in Egypt, with a special interest in assessing their zoonotic potential. The main strengths of the survey include (i) the use of a large sample size, (ii) the coverage of three different geographical regions, (iii) the use of highly sensitive PCRs as screening methods, and (iv) the use of specific PCR protocols for genotyping/subtyping purposes. Molecular information on the investigated protist species is particularly scarce in Egyptian animal populations in general and dogs and cats in particular. The study expands and complements information already provided by our research team on the epidemiology of enteric protists of public veterinary relevance in livestock species including buffaloes, cattle, and dromedary camels (55, 56).

Cryptosporidium spp. infections in Egyptian canine populations have been previously reported in the range of 2%–50% (Table 1) by conventional microscopy examination. In the only molecular-based study conducted to date, a prevalence of 24% (12/50) was found in household dogs in Sharkia Province (26). These highly variable prevalence rates are likely the reflection of changing epidemiological scenarios with differences in reservoir host populations, parasite´ strains, environmental and care conditions, sources of infection, and transmission pathways. This seems to be also the case of the present study, were Cryptosporidium spp. were detected at low rates (2%) in dogs from Giza governorate, but not in dogs from Gharbeya governorate. Our molecular analyses confirmed the presence of canine-adapted C. canis as the only Cryptosporidium species circulating in the surveyed dog population. This is in contrast with the evidence available in the country, where C. parvum was previously identified in five household dogs in Sharkia Province (26), and in two puppies with diarrhea in Qalubiya governorate (30). An asset of the present study is the use of a recently developed subtyping tool based on the amplification of partial sequences of the highly variable gp60 gene to ascertain subtype families within C. canis (42). This methodology has allowed the identification of nine (XXa to XXi) subtype families of C. canis in a variety of animal hosts including dogs, foxes, minks, and racoon dogs, in addition to humans (42, 57, 58). The finding of C. canis in a number of human isolates suggests that this species might represent a public health concern for vulnerable populations such as children and immunocompromised individuals. In our study we managed to subtype one of the four C. canis isolates, which was assigned to novel subtype XXe2. This result contributes to expand our knowledge on the genetic diversity and host range of this Cryptosporidium species.

Our study represents the first PCR-based description of Cryptosporidium infections in domestic cats in Africa. Using molecular methods, feline cryptosporidiosis has been documented at prevalence rates of 8%–13% in the Americas including United States, of 1%–12% in Asia (mainly China), of 2%–10% in Australia, and of 5%–7% in Europe (14). The prevalence rate found in our feline population (6.0%) falls well within the range of those figures reported globally. Our molecular analyses provided interesting data. Unexpectedly, C. parvum was far more prevalently found than feline-adapted C. felis (83.3% vs. 16.7%). This is in spite of Cryptosporidium felis is known to be the dominant species in cats globally (14), although other Cryptosporidium species including C. parvum (59, 60), C. muris (61, 62), and C. ryanae (62) have been sporadically reported in domestic cats. Our sequence analyses revealed that all five C. parvum isolates corresponded to genetic variants of the bovine genotype of the parasite (63), known to have a loose host specificity and therefore a clear zoonotic potential (64). The bovine genotype of C. parvum accounts for 43%–100% of confirmed bovine cryptosporidiosis cases in cattle in Egypt (65–69). We hypothesize that the high proportion of feline infections by C. parvum detected in our feline population can be the result of cross-species transmission between cattle and domestic cats sharing habitats under high infection pressure conditions. Examples of such events have been reported in other studies (70).

Available microscopy-based epidemiological data have demonstrated the occurrence of G. duodenalis in 1%–32% of the canine populations investigated in Egypt (Table 1). None of these studies used PCR as screening method. In the present survey we found a higher G. duodenalis prevalence of 38.5%. This was an expected result, as qPCR has a superior diagnostic performance compared with microscopy (13). As in the case of Cryptosporidium canine infections, large differences in G. duodenalis prevalence rates were observed between geographical areas, with the bulk of the infections (99%) coming from the Giza governorate. Potential explanations for this finding are the higher number of samples collected in this governorate compared with those from Gharbeya, differences in animal care and wellbeing standards and even local variations in the epidemiology of the parasite including sources of infection and transmission pathways (71). Remarkably, 56% (47/84) of the canine cases of giardiasis had qPCR C_T values > 32, suggestive of light infections. This fact might also explain the limited number of G. duodenalis isolates successfully subtyped at the gdh and/or bg loci. The four G. duodenalis isolates characterized corresponded to canine-adapted assemblages C and D. These results are in agreement with those reporting assemblage D in four microscopy-positive household dogs visiting private pet clinics in different Egyptian governorates (35).

In the only available survey investigating the presence of G. duodenalis in domestic cats in Egypt, the presence of the parasite was identified by conventional microscopy in 14.8% of the animals examined (31). The global prevalence of feline giardiasis has been estimated at 2.3% (5,807/248,195) in a systematic review and meta-analysis of prevalence studies (n = 68) from stool samples using a variety of diagnostic methods including light microscopy, IFA, ELISA, and PCR (13). We found a much higher prevalence of 32.1% using a highly sensitive qPCR assay. Unfortunately, 90.7% (39/43) of the feline samples positive for G. duodenalis by this method yielded C_T values > 32, precluding us to determine the subtype of these isolates at the gdh, bg, and/or tpi loci.

In this study, E. bieneusi and Blastocystis sp. were undetected in the investigated canine and feline populations. These results are in contrast with those previously reported in Egypt. For instance, microsporidial spores were identified by microscopy examination of stained smears in 33.3 and 23.1% of canine and feline fecal samples, respectively (37). Subsequent nucleotide sequence analyses confirmed the presence of E. bieneusi and E. intestinalis in these host species. On the other side, Blastocystis sp. colonization/infection has been detected at low rates in domestic dogs by conventional microscopy (3.1%) and cats by PCR (2.6%) (28, 38), although other surveys failed to identify the presence of the protist using culture and PCR methods (38, 39).

Taking together, molecular subtyping data generated in the present study indicate that domestic dogs and cats are primarily infected with host-adapted species including C. canis and G. duodenalis assemblages C and D in the case of dogs and C. felis in the case of cats. These genetic variants are considered of limited, but by no means negligible, zoonotic potential, as all of them have been sporadically found in human cases of giardiasis and cryptosporidiosis (10, 64, 72). The exception of this general rule is the unusual high proportion of zoonotic C. parvum infections detected in cats, a finding that represents a public health concern and should be further investigated. It should be noted that, out of 56 molecular studies in African countries, C. parvum ranked second after C. hominis as the most prevalent Cryptosporidium species circulating in humans (71).

Our results showed that canine and feline populations from Gharbeya governorate harbored lower parasitic prevalence rates than their counterparts from Dakahlia and Giza governorates. These discrepancies might be attributed to differences in sample size or the sanitary conditions under which the animals were kept (71). This study has some methodological limitations that should be taken into consideration when interpreting the obtained results and the conclusions reached. First, sample size varied among sampling areas, potentially biasing the accuracy of the statistical analyses conducted. Second, sample storage and transportation conditions might have altered the quality and quantity of available parasitic DNA, compromising the performance of the molecular methods used. Finally but not least, suboptimal amount of parasitic DNA might have hampered the PCR methods used for subtyping purposes, all of them based on the amplification of single copy genes including gdh, bg, and tpi (for G. duodenalis) or gp60 (for Cryptosporidium spp.).

This is one of the few molecular-based epidemiological surveys assessing the role of domestic dogs and cats as potential reservoirs of human infections by diarrhea-causing enteric protist parasites of public veterinary health relevance in Egypt. The main contribution of the study to the field include: (i) the confirmation that G. duodenalis, and to a lesser extent, Cryptosporidium spp. infections are common in household dogs and cats, (ii) the first description of the occurrence and molecular diversity of Cryptosporidium spp. infections in domestic cats in Africa, (iii) dogs are infected by canine-adapted pathogens, but cats carried an unusual high proportion of infections with zoonotic C. parvum that might represent a public health concern, (iv) the first description of C. canis subtype XXe2, and (v) the confirmation that strict carnivores such as dogs and cats are poor host species for Blastocystis sp. Molecular epidemiological data presented here might be useful for assenting health authorities and policy makers in designing and implementing effective intervention strategies against these zoonotic pathogens in Egypt. Simple and easy to implement measures include adequate hygiene practices (adequate canine and feline waste disposal, regular hand washing) and routine veterinary care are essential to prevent enteric parasite infections and minimize the risk of zoonotic transmission. Further research should explore the role of other domestic and wildlife species as potential reservoirs of human infections by enteric protists.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/nuccore; OQ778995–OQ779000, OQ787086, and OQ787087–OQ787091.

This study was approved by the Research Ethics Committee of the Faculty of Veterinary Medicine, Sohag University (Egypt) on 01.12.2019. Written informed consent was obtained from the owners for the participation of their animals in this study.

EE, AGa, AA-O, AGo, SM, YM, and ME collected the samples. EE, PCK, CH-C, and BB conducted laboratory experiments. PCK, AD, and LX conducted sequence analyses. EE, AD, JA, and CH-C conducted statistical analyses. MM and EH secured the funding for conducting sampling and experimental work. EE, DG-B, and DC designed and supervised the experiments. EE and DC wrote and prepared the original draft. EE, AGa, SM, AD, DG-B, LX, and DC wrote, reviewed, and edited the manuscript. All authors contributed to the article and approved the submitted version.

This study was partially funded by the Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness under project PI19CIII/00029. This study was supported by Researchers Supporting Project number (RSPD2023R655), King Saud University, Riyadh, Saudi Arabia.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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