Molecular detection of Cryptosporidium spp. and Microsporidia in human and animal stool samples

doi:10.21203/rs.3.rs-3338204/v1

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Molecular detection of Cryptosporidium spp. and Microsporidia in human and animal stool samples

https://doi.org/10.21203/rs.3.rs-3338204/v1

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Purpose Cryptosporidium spp. and Enterocytozoon bieneusi, are opportunistic microorganisms having remarkable zoonotic transmission potential due to their capacity to infect humans and animals. The aim of this study is to evaluate the prevalence of these microorganisms in stool samples from animal and human origin.

Methods Three hundred and sixty nine stool samples (205 from human patients with diarrhea and 164 of animal origin) were included in the study. Cryptosporidium spp. and E. bieneusi presence was determined by multiplex nested PCR. Positive results were analyzed by Sanger sequencing of the amplicon, using BLASTN and ClustalX software, to confirm identification.

Results Cryptosporidium spp. was found in 0.97% and 4.26% of human and animal samples, respectively (1 cat; 3 dogs, 1 goat and 1 Russian hamster). E. bieneusi was detected on human and animal stools (4 dogs and 1 pig), in the 6.82 % and 3.05% of the samples respectively.

Conclusion This study evidences the presence of Cryptosporidium spp. and E. bieneusi in human and animal samples from different species, and has as most interesting findings the detection of Cryptosporidium spp in pets that are not usually included in this type of studies, such as rodents and the identification of E. bieneusi in patients with diarrhea of different age groups and without underlying disease.

Cryptosporidium spp.

Enterocytozoon bieneusi

pets

farm animals

humans

stool

Cryptosporidium spp. is a protozoan parasite, within the Phylum Apicomplexa, that infects the microvilli of the gastrointestinal epithelium of a wide range of vertebrates, including humans [1]. Profuse watery diarrhea has become the more common clinical sign. Moreover, some extraintestinal cryptosporidiosis have been registered, where lungs, hepatobiliary system and pancreas were mainly affected [2]. Cryptoporidiosis have been described in immunocompromised as well as immunocompetent people, and large-scale outbreaks have been reported [3, 4], mainly associated with recreational waters, animal contact, environmental contact, person-to-person spread and contaminated food or water [3, 5–7]. While cryptosporidiosis is recognized as being among the most common causes of human parasitic diarrhea in the world, there is currently limited knowledge on Cryptosporidium infection mechanisms, incomplete codification of diagnostic methods, and a need for additional therapeutic options [8].

Microsporidia are a ubiquitous group of spore-forming obligate intracellular microorganisms that infect a wide range of vertebrates and invertebrates [9]. They belong to phylum Microsporidia within the kingdom Fungi.

However, at taxonomic level there are some contradictions, remains it in doubt. More than 220 genera, where are included further 1,700 species, have been described until nowadays. Nine of these genera (Encephalitozoon, Enterocytozoon, Pleistophora, Trachipleistophora, Vittaforma, Anncaliia, Endoreticulatus, Tubulinosema y Microsporidium) [10] and seventeen species are related to human infection, becoming E.bieneusi and E. intestinalis the most common between them [11]. Concretely, these species are responsible of an extensive variety of systemic and non-systemic diseases. Although chronic diarrhea is the clinical manifestation that best correlates with the infection due to these microorganisms, ocular, breathing, renal and nervous systems infections are also described [2, 5]. Since the discovery of the species E. bieneusi, the number of patients affected by this microorganism has increased considerably. As a result, microsporidia has become casual agents of intestinal and systematics infections in patients with human immunodeficiency virus (HIV) or other immunocompromised individuals. Additionally, the presence of Microsporidia has been described in feces of immunocompetent people [12]. Transmission occurs mainly through fecal-oral routes; accordingly, sources of infection usually are infected people or animals. Moreover, even food or water contaminated with spores could be an infection focus [13]. Several works show the zoonotic potential of microsporidia [13, 14].

Although Cryptosporidium spp. and Enterocytozoon bieneusi, are opportunistic microorganisms with potential for zoonotic transmission, information on their prevalence in pets and other animals in close contac with humans and their contribution as an etiological agent of diarrhea in humans (mainly due to the lack of routine diagnosis in clinical laboratories) is scarce. In Spain, there has been a remarkable increment in the number of pets owners in the last years (https://www.anfaac.org/datos-sectoriales/). In this study, we evaluate the prevalence of these microorganisms in stool samples from animal and human origin.

Ethical considerations

In the preparation of this study, the recommendations of the Cadiz Research Bioethics Committee for biomedical research studies were followed. The study protocol was approved by this committee.

Origin of specimens

A total of 164 feces of animal origin and 205 feces of human origin were collected during three years (2017, 2018 and 2022). The samples of animal origin refer to pets and farm animals collected in the province of Cádiz. Dogs, cats, cows, pigs and goats’ samples were collected by swab and chicken samples were recollected from cottages, in a collective way; doves feces were sampled directly from deposition places.

Human samples belong to surplus diarrhea samples. These samples were sent to the Clinical Microbiology laboratory of the Hospital Universitario Puerta del Mar for routine stool culture, including in the study all the samples received in the laboratory during one day of each month. All surplus diarrhea and animal samples included in the study were stored at -80ºC.

DNA extraction

After thawing the samples, ASL buffer for DNA extraction (QIAGEN GmbH, Hilden, Germany) was used, following the manufacturer's instructions. Briefly, 500 µl of ASL buffer was added to 15 µl of the stool sample or its suspension in sterile distilled water, the mix was vortexed for 20 seconds and incubated for 10 minutes at 95°C, and then centrifuged at 1,3000 for 5 minutes. The supernatant was used for DNA extraction, using the EZ1 automated extraction system (QIAGEN).

Multiplex nested PCR

For multiplex nested PCR, the method proposed by Rubio et al was performed [5]. In each run, positive controls (kindly provided by Dr. David Carmena, from the Parasitology Reference and Research Laboratory, National Center for Microbiology, Majadahonda, Madrid, Spain) and negative controls for Cryptosporidium sp. and E.bieneusi were included. Positive results were confirmed by Sanger sequencing of the amplicon, using BLASTN and ClustalX software to confirm identification.

Two hundred and five samples of human origin were analized. Epidemiological characteristics of the patients included in the study are presented in Table 1. 48.3% of the samples came from women, while samples from men accounted for 51.7% of the total. In addition, 5.8% of the samples came from oncologic patients, 3.9% from patients affected by chronic kidney diseases, 0.97% from patients diagnosed with chronic hepatitis and 0.97% from patients diagnosed with HIV.

Table 1

Number of hospitalized and non-hospitalized patients by age range.
Age range	Outpatient N (%)	Inpatient N (%)	Total (N)
0–14	32 (19.4)	2 (5.0)	34
15–40	44 (26.6)	2 (5.0)	46
41–65	59 (35.7)	14 (35.0)	73
> 66	30 (18.1)	22 (55.0)	52
Total (N)	165	40	205

Besides, 164 samples from different species of animal origin were collected and analyzed (Table 2). The 50% of these samples came from dogs, been the most representative species, followed by birds and cats, which represented the 16.5% y 14% of the samples respectively. Samples from rodents represented the 5.5% from the total, and the remaining (14%) from different farm animals.

Table 2

Main epidemilogical features of the animals populations investigated in the present survey
	Dog (n = 83)	Cat (n = 23)	Rodents^a (n = 9)	Birds^b (n = 27)	Others^c (n = 22)
Pets	100%	100%	100%	22.2%
Farm				77.7%	100%
^a6 rabbits, 2 hamster and 1 peruvean guinea pig
^b1 turtledove, 2 ducks, 1 turkey, 1 canary, 2 lovebirds, 1 parrot, 1 kestrel, 4 pigeons and 14 chickens
^c5 pigs, 6 goats, 11 cows

Cryptosporidium spp and Microsporidia detection in human feces samples.

Cryptosporidium spp was detected in 2 samples (0.97%), and E. bieneusi in 14 samples (6.82%) of 205 human feces. Epidemiological and clinical characteristics of the patients with positive results are showed in Table 3.

Table 3

Positive samples for *Enterocytozoon bieneusi* and *Cryptosporidium spp*. in faecal samples from human origin.
ID	Sex	Age (years)	Underlying disease	Other enteropathogens	Origin	Microorganism
5	Female	61	Lymphoma		Inpatient	Cryptosporidium spp
20	Male	22			Outpatient	Encephalitozoon bieneusi
26	Male	80			Inpatient	Encephalitozoon bieneusi
42	Female	24			Outpatient	Encephalitozoon bieneusi
101	Male	0 (1 month)			Outpatient	Encephalitozoon bieneusi
106	Female	57			Outpatient	Encephalitozoon bieneusi
111	Female	37			Outpatient	Encephalitozoon bieneusi
115	Female	21			Outpatient	Encephalitozoon bieneusi
118	Male	10		Campylobacter sp.	Outpatient	Encephalitozoon bieneusi
120	Female	7		Campylobacter sp.	Outpatient	Encephalitozoon bieneusi
125	Female	32			Outpatient	Encephalitozoon bieneusi
128	Male	89	Melanoma		Outpatient	Encephalitozoon bieneusi
129	Male	63	Lung adenocarcinoma		Outpatient	Encephalitozoon bieneusi
131	Female	42			Outpatient	Encephalitozoon bieneusi
180	Male	6			Outpatient	Encephalitozoon bieneusi
186	Male	59	Metastatic Colon Adenocarcinoma		Inpatient	Cryptosporidium spp

Cryptosporidium spp and Microsporidia detection in animals feces samples.

Cryptosporidium spp. and E.bieneusi were detected in 4.26% and 3.05% of the samples of animal origin, respectively. Epidemiological and clinical characteristics of animals with positive results are presented in Table 4.

Table 4

Positive samples for *Enterocytozoon bieneusi* and *Cryptosporidium spp*. in faecal samples from animal origin.
ID	Host	Breed	Age	Microorganism	Diarrhea	Typeof population
17	Cat	Common european	3 months	Cryptosporidium spp	yes	Pet
26	Dog	English setter	10 years	Encephalitozoon bieneusi	no	Pet
29	Dog	Yorkshire	16 months	Cryptosporidium spp	no	Pet
67	Pig	unknown	Breeding	Cryptosporidium spp	unknown	Farm
68	Pig	unknown	Breeding	Encephalitozoon bieneusi	unknown	Farm
69	Goat	unknown	Breeding	Cryptosporidium spp	unknown	Farm
70	Dog	Hungarian Shorthaired Pointer	1 year	Cryptosporidium spp	no	Pet
75	Dog	Mongrel	4 years	Encephalitozoon bieneusi	no	Pet
94	Dog	Golden Retriever	14 months	Encephalitozoon bieneusi	no	Pet
96	Dog	Winery	15 years	Cryptosporidium spp	no	Pet
123	Hamster	Russian	1 year	Cryptosporidium spp	no	Pet
135	Dog	Mongrel	2 years	Encephalitozoon bieneusi	no	Pet

In Spain, there had been a remarkable increment in the number of pets owners. In 2021, the number of dogs and cats has been estimated around 9.3 and 5.8 million, respectively(https://www.anfaac.org/datos-sectoriales/).

Despite of companionship from these animals, which provide a clear benefit for people, pets can carry diseases that spread between them and humans, pretense a potential threat to public health [15].

Our study evidenced the presence of Cryptosporidium spp. and E. bieneusi in humans and domestic and farm animals. These animals are in a close contact with human populations, and could be an important zoonotic factor for infection and a possible source of environmental contamination [16].

We have detected Cryptosporidium spp. in only two samples from human feces, corresponding to adult oncologic patients. However, no positive samples from pediatric feces where detected, according to a previous study performed in the community of Madrid, where the presence of Cryptosporidium spp. in children from 1 to 16 years-old was very low (0.9%) (13/1512) [17]. In a study carried out in Brazil, including children with underlying diseases such as diarrhea, cancer, HIV and malnutrition, the prevalence was 2.2% (14/626) in this age range [18]. In samples of animal origin, we detected the presence of Cryptosporidium spp. in a cat suffering diarrhea (4.34%; 1/23) as well as in three dogs (3.61%; 3/83). A recent study carried out in Poland showed similar results, describing the presence of this microorganism in two felines, also affected with diarrhea, (n = 101; 2%) and nine asymptomatic dogs (n = 264; 3,4%) [19]. This study emphasizes the fact of the presence of diarrhea in both felines, just as it has been described in our study. Moreover, another study performed in Austria showed a prevalence of 1.7% (37/298) when cats where analyzed [20]. A study carried out in 2010, highlighted the presence of these parasites (9%) in fecal samples from dogs and cats took from public parks in Madrid [14].

In addition, Cryptosporidium spp. was detected in two farm animals: a lactating pig, (20%;1/5) and a goat (16.66%; 1/6). A similar study carried out on farms in China found a prevalence in lactating pigs of 2.7% (8/299) [21]. The prevalence of this microorganism in goats is high, such as revealed a study carried out in Ecuador, where the 10.49% of the goats were infected by Cryptosporidium spp. [22]. A similar study described a prevalence of 14.3% (1/7) in different farms located in the north of Spain (Galicia) [23].

In our study, it has been described the presence of Cryptosporidium spp. for the first time in a Russian hamster, the only one that was included. A recent research including these rodents, in China, [24] described an incidence of 39.32% in this species (138/351). However, there is a lack of information focused on this kind of rodents, and it should be interesting to include rodents in future studies, due to its possible implication in human health, as they are usually found as pets.

In our study, when infections by E. bieneusi were analyzed, results revealed interesting data. In the case of infection by E. bieneusi, the percentage of infected patients aged 14 years and older was lower than in pediatric patients (5.8% vs 11.7%), and included two adult oncologic patients (2/171). One research carried out in Egypt described a prevalence of 4.6% (27/585) in pediatric patients and remarks that children from rural zone, who usually are more in contact with animals than children from urban areas, have a higher infection rate [25]. This fact is

corroborated by a study carried out in China, where a higher infection rate by E. bieneusi in rural compared to urban areas was estimated [26]. Our study included children from urban areas from two cities with more than 95,000 citizens.

It has been showed that species of microsporidia constitute one of the more important opportunistic pathogens, due to its ability to cause grave disease after infection, especially in immunocompromised patients [27]. Therefore, clinical course of microsporidiosis depends on host immune state, where the infections occurs and the microsporidium specie implicated [28]. The number of non-infected patients by HIV who present other inmunodeficient problems, as transplanted or oncologic patients, have been also increased [5].

A recent study carried out in Spain showed the relation between colon cancer and Microsporidia infection, as revealed the analysis of tissue samples [29]. The presence of healthy carriers has also been documented, that clearly shows the presence of Microsporidia in feces of immunocompetent patients and that microsporidiosis may not be linked to any clinic in healthy populations, detecting individuals infected with E.bieneusi totally asymptomatic [12].

Remarkably, in our study, the majority of patients with E. bieneusi did not have any serious underlying disease, neither other enteropathogens causing diarrhea were found. However, it would be necessary more studies to clarify the need of including the determination of this microorganism in the routine study of diarrhea. It could be specially interesting when no other enteropathogens are found in the usual determinations.

Regarding the prevalence of E. bieneusi in different animals, our results revealed its presence, mainly in dogs (4.81%; 4/83). Several studies carried out in Spain show that the prevalence of this microorganism in dogs varies considerably, with percentages between 0.8% and 11.7% [11, 15, 23]. These studies included pet as well as animals from the street, being the last group the biggest carrier of the microorganism. In Chine, other studies revealed an incidence of 6.74% (18/2067), and most infections occur in males, younger than one year old and collected from a pet market [30]. Other study carried out in Australia showed an incidence of 4.4% (15/342) [31]. However, in a recent study carried out in Portugal, no positive samples for E. bieneusi were found in 46 fecal samples [32]. In addition, in our study we detected the presence of E. bieneusi in feces of a pig (20%; 1/5). According to these results, a previous study carried out in the South of Spain (Cordoba) revealed an incidence of 22.6% in these animals [33].

In conclusion, this study evidences the presence of Cryptosporidium spp. and E. bieneusi in human and animal samples, and has as its most interesting findings, on the one hand, the detection of Cryptosporidium spp in pets that are not usually included in this type of studies, such as rodents and, on the other hand, the identification of E. bieneusi in patients with diarrhea of different age groups and without underlying disease. It would be desirable to carry out further studies on these two aspects, analyzing the risk of zoonotic transmission of certain types of pets and the convenience of including the determination of E. bieneusi in the routine study of diarrhea.

Acknowledgments

The authors are indebted to Dr. David Carmena of Parasitology Reference and Research Laboratory, National Centre for Microbiology, Majadahonda, Madrid, Spain, for his invaluable help.

Author Contributions

Conceived and designed the experiments: FG TG MI. Performed the experiments: FG TG. Analyzed the data: FG TG. Contributed reagents/materials/analysis tools: FG TG. Wrote the paper: FG MI TG. Manuscript review: FG MI TG

Conflict of interest

The authors have no competing interests to declare that are relevant to the content of this article.

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Molecular detection of Cryptosporidium spp. and Microsporidia in human and animal stool samples

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Abstract

Introduction

Material and Methods

Ethical considerations

Origin of specimens

DNA extraction

Multiplex nested PCR

Results

Discussion

Declarations

Acknowledgments

Author Contributions

Conflict of interest

References

Status:

Journal Publication

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