Identification of Cryptosporidium species , Enterocytozoon bieneusi genotypes, and Giardia duodenalis assemblages in birds in Henan, China

Domesticated, wild, and migratory birds have been known to transmit diseases such as diarrhea in humans and other animals, but studies specifically on the zoonotic pathogens Cryptosporidium spp., Enterocytozoon bieneusi , and Giardia duodenalis in birds in Henan Province, China are lacking. Hence, this study sought to characterize the prevalence of these pathogens, and to identify the different species of Cryptosporidium and their phylogenetic relationships, the genotypes of E. bieneusi , and the assemblages of G. duodenalis , in birds in the province. Fresh fecal samples were collected from birds in parks and pet in China and were screened for the presence of the pathogens using nest-PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene and the internal transcribed spacer (ITS) gene. from , ,


Conclusions
Our findings indicate that the aforementioned species of birds in Henan, China could be a source of zoonotic pathogens, such as C. meleagridis, C. andersoni, C. parvum, E. bieneusi genotype Peru6, and G. duodenalis assemblage E, that cause diseases in humans.

Background
Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis are zoonotic pathogens that cause acute or chronic diarrhea, vomiting, and respiratory ailments in both humans and animals via fecal-oral transmission [1].
Cryptosporidiosis is a parasitic disease in caged, domesticated, and wild birds [2].
Currently, four Cryptosporidium species, including C. meleagridis, C. baileyi, C. galli, and C. avium, and several genotypes including the avian genotypes I-V, Eurasian woodcock genotype, black duck genotype and goose genotypes I-V have been detected in birds through sequence analysis of the SSU rRNA gene [2][3][4]. Moreover, birds infected with C. hominis, C. serpentis, C. andersoni, and the muskrat genotype I and with the zoonotic pathogens C. parvum, C. muris, C. andersoni and C. meleagridis have also been reported [5].
The pervasive obligate intracellular eukaryotic fungi E. bieneusi infects humans and various animals, including birds, fish, insects, amphibians, and mammals, specifically dogs, cats, pigs, rabbits, and sheep. More than 300 genotypes of E.
bieneusi have been reported based on differences in ITS gene sequences [6]. Based on phylogenetic analysis, E. bieneusi could be clustered into nine groups, one of which is zoonotic group 1 that can infect humans and animals. The genotypes of E.
Giardiasis is another parasitic disease caused by six Giardia species, which are zoonotic pathogens that have been differentiated from others based on morphological, host-source, and electron microscopic nuances. As far as public health is concerned, the most zoonotic among these species is G. duodenalis. Based on genetic analyses, G. duodenalis consists of eight distinct assemblages from A to H, of which only the zoonotic assemblages A and B and nonzoonotic assemblages D and F have been found in birds [10][11].
Birds that are in close contact with humans and those that migrate have been known to play a consequential role in the transmission and spread of zoonotic parasites [1]. Nevertheless, there have only been a few studies on the epidemiological and genetic characteristics of Cryptosporidium spp., E. bieneusi and G. duodenalis in birds in Henan Province, China. For this reason, this study aimed to primarily evaluate the prevalence of the pathogens and specifically identify, through nucleotide sequencing, the different species of Cryptosporidium and their phylogenetic relationships, the genotypes of E. bieneusi, and the assemblages of G. duodenalis present in birds in Henan, China.

Fecal sample collection
A total of 1,005 fresh fecal samples were collected from 32 species of birds (Table   1) in parks and pet shops in seven areas in Henan Province, China from July 2018 to May 2019 ( Figure 1). Approximately 1-10 birds of the same species were caged in groups, and only one specimen was collected from each cage. Using sterile gloves, the specimens were briefly collected from the ground or cages. The time of collection, location, taxonomic identification, deworming conditions, feeding habits, and the shape of the feces of the birds were recorded. About 200-300 mg of each fecal sample was dispensed into a 2.5-ml centrifuge tube and was stored at 4°C before DNA extraction. No mixed infection was observed among the samples.

DNA extraction and PCR amplification
Whole genomic DNA was extracted from each of the fecal samples using an EZNA Stool DNA Kit (Omega Bio-tek Inc., Norcross, GA), and according to the manufacturer's instructions it was eluted in 200 µl of elution buffer and was stored at -20°C until PCR amplification. All the extracted DNA was screened for the presence of Cryptosporidium spp . and G. duodenalis through the amplification of the small subunit ribosomal RNA (SSU rRNA) gene [12,13] and the presence of E.
bieneusi was established through the amplification of the internal transcribed spacer (ITS) region of the rRNA gene [14]. Negative and positive control samples were included in every PCR reaction. Secondary PCR products were subjected to electrophoresis in 1% agarose gel, they were stained with DNA Green (TIAND, Beijing, China) and were visualized with a UV transilluminator.

Sequence analysis and phylogenetic analysis of Cryptosporidium spp.
All the PCR-positive products were submitted to the Beijing Nuosai Biological Engineering Company for bidirectional sequencing using an ABI PRISM 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA). The nucleotide sequences obtained in this study were compared with the reference sequences downloaded from the GenBank database and were aligned using ClustalX 2.0 (http://www.clustal.org/).
The phylogenetic analysis of Cryptosporidium spp. based on the SSU rRNA gene was performed using software MEGA 7.0 (https://www.megasoftware.net/). Neighborjoining phylogenetic trees were constructed based on the evolutionary distances calculated using Kimura's two-parameter model, and the reliability of the trees was assessed through bootstrap analysis with 1,000 replicates.

Statistical analysis and nucleotide sequence accessioning
All statistical analyses were performed using SPSS 22.0. Statistical differences among the sampling areas, deworming conditions, and fecal shapes were evaluated using a Chi-square test. Values with p < 0.05 were considered significant, and a 95% confidence interval was determined. Representative nucleotide sequences generated in this study were deposited under accession numbers MN410718 to MN410725 to the GenBank database.

Cryptosporidium species and sequence analysis
Among the fecal samples that were found to be positive for Cryptosporidium spp., five species were further identified ( Figure 2). The most predominant among them was C. baileyi (10 out of 21 fecal samples, 47.62%), whose nucleotide sequences were detected in three crested mynas (Acridotheres cristatellus), two Java sparrows (Lonchura oryzivora), one Chinese hwamei (Garrulax canorus), two common quails (Coturnix coturnix), and two Chinese grosbeaks (Eophona migratoria) and were found to be 100% homologous with a sequence (KT151550) identified in quail in Iraq. Another species, C. meleagridis (4/21, 19.05%), was identified in three parrots and in one crested myna. Its sequences were 100% homologous and were identical to that of an immunocompetent patient (MK311181) from Poland. The sequences of C. galli (5/21, 23.81%) were detected in three white-eyes (Zosterops sp.), one zebra finch (Taeniopygia guttata) and one Chinese blackbird (Turdus mandarinus), and they were 99% homologous with a sequence (MG516766) identified in an ibis in Australia. The pathogen C. andersoni (1/21, 4.76%) was detected in one white-eye for the first time, and its sequence showed 100% homology with that of a human patient (KF826301) in China. The sequence of C. parvum (1/21, 4.76%) was found in a pigeon of family Columbidae and was found to be 100% homologous with a sequence (MK241967) detected in cattle from India.

E. bieneusi genotypes and sequence analysis
The sequences of E. bieneusi were found in ten European turtle doves (Streptopelia

G. duodenalis assemblages and sequence analysis
The pathogen G. duodenalis was identified in 12 parrots, three common hill mynas, 13 crested mynas, one Java sparrow, one white-eye, one black-throated laughingthrush and two other birds in Zhengzhou (6.85%). All the samples positive for G. duodenalis were identified to be from assemblage E and were 100% homologous with the isolate MH794176 from a sheep in China (Table 2).

Discussion
This study principally demonstrated the varying prevalence and genetic characteristics of Cryptosporidium spp., E. bieneusi, and G. duodenalis in birds in Henan Province, China. As far as we know, our molecular data on E. bieneusi and G. duodenalis in birds are the first to have been generated in the region.
Cryptosporidium spp. have been reported in many species of domesticated and wild birds. However, in this study, Cryptosporidium spp. showed a prevalence or infection rate of only 2.09%. This is higher than that reported in Poland (1.1%) [15] and Japan (0%) [16] but lower than that recorded in domesticated and wild birds in Italy (5.7%) [17], Malaysia (10%), Brazil (4.86%) [4], northwestern Spain (8.3%), Hungary (8.8%), Australia (6.3%), the United States (7.2%), Greece (13.0%), and Japan (9.1%), and particularly in China, it is lower than that documented for central China (8.1%) [18] and northeast China (11.1%) [19]. Molecular techniques were employed in this study to record the infection rate of Cryptosporidium spp., but through microscopic examination, Gu [20] found that the infection rate of Cryptosporidium in birds in Anhui Zoo was only 0.97%. Thus, using molecular techniques to detect Cryptosporidium may be more efficient and accurate than performing microscopic examination. The differences in infection rates between the current study and previous ones could be attributed to the differences in study area, methods, bird species, and bird feeding habits.
A total of five Cryptosporidium species were detected in birds in this study. The species C. baileyi, C. meleagridis, and C. galli are considered the most common pathogens in birds worldwide. This study was the first to identify C. baileyi in Chinese grosbeak and Chinese hwamei, C. meleagridis in crested myna, C. galli in zebra finch, Chinese blackbird, and a species of white-eye, and C. andersoni in a white-eye. Reboredo-Fernandez reported the detection of C. parvum in sparrowhawk and black-billed magpie (Pica hudsonia) in northwest Spain, but in the current study, C. parvum was found in a pigeon. Importantly, C. meleagridis, C. andersoni and C. parvum can infect humans. The results of this study indicate that the range of bird species hosting Cryptosporidium spp. could be wider, and the diversity of Cryptosporidium spp. acting as zoonotic pathogens could be greater than what was previously known.
The average infection rate (4.48%) of E. bieneusi documented in this study is lower than that recorded in Heilongjiang Province (22.2% in pet birds), Portugal (12.8% in pet birds), and Czech Republic (21.3% in exotic birds), but it is higher than that reported in Poland (1.4% in pigeons) [21] and Brazil (3.8% in exotic birds). Peru6, A and EbpA) have also been detected in humans. In this study, Peru6 was identified in 44 samples, suggesting that the genotype is dominant. Peru6 belongs to group 1 which is of notable zoonotic importance because it largely affects humans. The genotype has been isolated from HIV-infected patients in Peru, but not yet in China. However, Peru6 has already been found in pigeons, cranes, geese, and ducks in China. In this study, PtEb I was found for the first time in one pigeon in Luohe. The genotype has never been attributed to human infections.
Although G. duodenalis has been commonly detected in mammals, it has been scarcely reported in birds especially in China. This study is the first to identify the pathogen in birds in China. The infection rate (3.28%) recorded for G. duodenalis in this study is lower than that documented in Italy (4.3% in pet birds) [17], Spain (8.3% in aquatic birds) [24] and Japan (16.1% in pet birds). On the contrary, the prevalence of G. duodenalis that this study found in pet birds is higher than that in Brazil (1.1% in captive birds), northwest Spain (2.1% in wild birds) [25], Poland (2.2% in captive birds), and Japan (1.7% in zoo birds).
In this study, all the samples positive for G. duodenalis assemblage E were collected in Zhengzhou. It was previously reported that G. duodenalis assemblage E mainly infects artiodactyls, but in this study, G. duodenalis assemblage E was found in birds, probably because their food or water was contaminated with the pathogen.
There have been occasional reports of G. duodenalis assemblage E in the human body [26], indicating that G. duodenalis assemblage E is a zoonotic risk.

Conclusions
Overall, this study reported the occurrence and molecular diversity of Cryptosporidium spp., E. bieneusi, and G. duodenalis in birds in Henan Province, China. Our findings suggest that these pathogens infect a diversity of avian species, notably including those that were found to be positive for these pathogens for the first time. The detection of C. meleagridis, C. andersoni, C. parvum, E. bieneusi genotype Peru 6 and G. duodenum assemblage E in birds suggests that exposure to these birds pose a threat to human health. Most of the birds in which the pathogens were detected were ostensibly healthy, and they appeared to be asymptomatic carriers of the parasites. Pet owners and animal keepers who are responsible for the regular maintenance and feeding of birds are the most vulnerable to zoonotic infection. In addition, birds can travel long distances and could be important for spreading the pathogens. Hence, the potential mechanisms underlying the zoonotic transmission of diseases between birds and humans should be further investigated.

Availability of date and material
All date generated or analysed during this study are included in this published article. Sequence were submitted to the GenBank database under the accession number MN410718-MN410725.

Author contribution
fL and LZ designed the study. ML and RC collected and analyzed the specimens. RC, JL, and CB analyzed the date. RC, JD and LZ wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate
This study protocol was reviewed and approved by the research ethics committee of Henan Agricultural University (Zhengzhou city, China). Before beginning this study, we contacted the owners or managers of the animals and acquired their permission.
No birds were hurt during sample collection.    Phylogenetic relationships of Cryptosporidium species identified in this study and other know