Molecular epidemiology and genotype/subtype distribution of Blastocystis sp., Enterocytozoon bieneusi, and Encephalitozoon spp. in livestock: concern for emerging zoonotic infections

Intestinal parasitic infections have high prevalence rate in many regions especially in developing countries. The aim of this study was to determine the presence and genotype/subtype of some intestinal protozoa in livestock in Iran. Stool samples were collected from cattle, sheep, chickens, and horses. The presence of targeted parasites was evaluated using real-time PCR. Genotyping/subtyping of positive samples was characterized using sequencing of the ITS and barcoding region, respectively. Blastocystis sp., 27.7% (48/173) and Enterocytozoon bieneusi 26.0% (45/173) were the most frequent protozoa followed by Encephalitozoon spp., 0.57% (1/173). Cryptosporidium spp. were not detected among samples. Encephalitozoon spp., was detected only in chickens 2.2% (1/45). A statistically correlation was seen between animals and the prevalence of targeted protozoa. E. bieneusi genotypes I (9/38; 23.68%), BEB6 (22/38; 57.89%), D (6/38; 15.79%), and horse1 (1/38; 2.63%) were detected among samples. A statistically significant correlation was seen between the genotypes and animals (P ≤ 0.05). Blastocystis sp., ST1 (1/45; 2.22%), ST5 3/45; 6.66%), ST7 (1/45; 2.22%), ST10 (24/45; 53.33%), and ST14 (16/45; 35.55%) were characterized among samples. There was no significant correlation between certain subtypes and animals (P = 0.173). The presence of zoonotic potential genotypes of E. bieneusi in animals and zoonotic potential subtypes ST1 and ST7 among our samples provide a clue about the transmission dynamic of E. bieneusi and Blastocystis sp. between animals–animals and humans–animals.

Infections caused by intestinal parasites are still one of the important public health problems in the world. A wide range of helminths and protozoa can infect or colonize the gastrointestinal tract of humans and animals. The enteric protozoa such as Cryptosporidium spp., Enterocytozoon bieneusi, and Encephalitozoon spp., are of the most important zoonotic parasites causing diarrhea in humans 1 , which infect a wide range of domesticated and wild animals, as well 2 . In addition, Blastocystis sp. is the a prevalent protist, which its pathogenic role is still under question 3 .
These microorganisms are typically transmitted through several routes, such as direct contact with infected persons (anthroponotic transmission) or animals (zoonotic transmission), and ingestion of infective cyst/ oocyst/ spore through contaminated water or food 4,5 . Asymptomatic infections due to aforementioned parasites are mostly reported from immunocompetent subjects; however, a broad range of clinical manifestations like chronic diarrhea, nausea, weight loss, vomiting, dysentery, and fever have been recorded from children, travelers, and the elderly individuals. In general, the clinical symptoms in immunocompromised individuals, especially in HIV + / AIDS patients are more severe 6,7 .   Figs. 2, 3c). Furthermore, the prevalence rate of Encephalitozoon sp. in chicken was 2.2% (1/45) (Table 1). No statistical significant association was evidenced between the presence of Encephalitozoon sp. and the types of animal (P = 0.595). In addition, no cases of Cryptosporidium spp. were detected by real-time PCR. E. bieneusi genotyping and phylogenetic analysis. The ITS fragment of the ribosomal RNA (rRNA) gene was successfully amplified among 38/45 (84.44%) of real-time PCR-positive samples. All amplified samples  Table 5). The phylogenetic analysis showed that all subtypes were clearly separated based on the currently known subtypes. The phylogenetic tree also revealed that there was no separation based on the hosts. In addition, similar subtypes, which were isolated from different hosts, were clustered together with bootstrap support ranging from 80 to 99% (Fig. 2).

Discussion
Blastocystis sp., microsporidia, and Cryptosporidium spp., are among protozoa, which may be zoonotically transmitted to humans. In the current study 46.2% of samples were detected positive for selected parasites using real-time PCR. This prevalence rate is similar to previous reports from Belgium 20 , Canada 21 , France 22 , China 12 , but is higher than another report from China (25.6%) 23 . Although evaluated pathogens between our study and most of indicated reports are similar, it seems that method of evaluation has critical role in true estimation of the prevalence. Actually, in the study performed by Yu et. al. (2018) 23 , parasitological techniques were used to detect parasites while molecular genotyping was performed for only those samples, which were positive for Giardia and Cryptosporidium; therefore, a lower prevalence rate was expectable. In this regard, Incani et al., 24 investigated the prevalence of intestinal parasites in a rural community and compared the results with microscopy, and concluded that real-time PCR was a more sensitive technique, although microscopy could be advisable, particularly in cases without molecular tests. E. bieneusi is a prevalent microorganism in humans and animals. Increasing reports suggest the importance of zoonotic transmission of E. bieneusi due to the low host-specificity of most of its genotypes 18 . In the current study, E. bieneusi was detected from 34.4% (11/32), 25.7% (18/70), 28.9% (13/45), and 11.5% (3/26) of cattle, sheep, chickens, and horses, respectively, with an overall prevalence rate 26.0% (45/173). The prevalence of E. bieneusi in cattle is higher than previous reports from China 15,25,26 , Thailand 27 , Turkey 28 , Brazil 29 , Australia 30 , and the United States (USA) 31 , but it is in the line of reports from the USA 32 and China 33,34 . The genotypes D, hoerse1, I, and BEB6 were characterized in the current study that are categorized as groups 1a, 1e, 2b, and 2c, respectively. These genotypes were all or individually reported in studies from the USA 31,32,35 , Argentina 36 , Germany 37 , Australia 30 , Thailand 27 , and many studies from China 25,34,[38][39][40] . The genotype D is the most frequently reported genotype from humans and broad range of domesticated and wild animals 7,41-43 and thought to be a high zoonotic potential genotype with worldwide distribution. The genotype I is one of the most prevalent genotypes in cattle, which together with the genotype BEB6, were reported from humans 44,45 , as well. These genotypes are categorized among the genotypes with low level of host specificity and increasing zoonotic concerns 18,46 . In contrast to the genotype I, which is frequently reported from cattle and thought to be a cattle genotype, the genotype BEB6 is common in sheep and was suggested that this genotype has been probably adapted to cattle during years 46,47 .
The prevalence of E. bieneusi in sheep is in line of some studies from China 48,49 , higher than studies from Brazil 29 , Ethiopia 50 , and lower than reports from Sweden 47 and China 39,51 . In the line of our study, the genotype BEB6 is the most prevalent genotype reported from sheep 47,50,51 . However, this genotype represents low level of Table 4. The prevalence of Blastocystis sp., subtype in studied animals.  www.nature.com/scientificreports/ bieneusi was detected from 28.9% of chicken samples, which is higher than previous reports in the world. The reason for this observation could be related to the method of detection. Actually, in the current study, real-time PCR was employed to detect E. bieneusi, which has higher sensitivity compared to conventional PCR. The genotype D, BEB6, and I were characterized in chicken samples. As mentioned above, these genotypes show low level of host specificity and have been reported from broad range of animals 18 . The genotype D was reported from chickens in a study conducted by Cao et al. (2020) 56 , and is known as the most prevalent genotypes in the world. Many studies in Iran reported this genotype from humans 7,43 , wastewater 58 , vegetables 58 , and wild and domesticated animals 13,42,59,60 , which implies the cross-transmissibility of this genotype between humans and animals and the importance of zoonotic transmission of the genotype D in Iran. The presence of the genotypes BEB6 and I in chicken samples indicates high host-multiplicity and -adaptation of these genotypes.

Total (%) ST1 (%) ST5 (%) ST7 (%) ST10 (%) ST14 (%)
Reports of E. bieneusi in horses are not too much. In current study, 11.5% of horses harbored E. bieneusi, which is close to the previous reports [61][62][63][64] , but lower than most of reports from China [65][66][67] . In Turkey, a country neighboring Iran, 18.7% of horses were detected positive for E. bieneusi 68 . E. bieneusi genotype horse1 thought to be a horse-specific genotype. This genotype was reported from horses in studies from Colombia 61 , Czech Republic 62,64 Algeria 63 , and China 65,66 . However, this genotype is categorized in group 1, which is known as zoonotic group and might be an emerging zoonotic genotype in Iran. In addition, the genotype BEB6 was previously reported from horses in Turkey 68 and China 65,67 . The presence of the genotype BEB6 in horses, chickens, cattle, and sheep in our study implies the non-host specificity of this genotype and high capability of the genotype BEB6 for adaptation in different hosts (Table 6).
Blastocystis sp., was the most prevalent protozoan among samples 27.7% (48/173). Blastocystis sp. is a protist, which is frequently reported from humans 95,96 and animals 97 . The prevalence rate of Blastocystis sp. in this study was higher than recent reports from Iran that indicated a rate of 14.98% 98 among cattle, sheep, and, poultry, and 17.5% 99 among dog and cat samples. Increasing evidence suggest the importance of animal to human transmission besides human to human transmission of Blastocystis sp. Until now, 17 genetic lineages (subtypes) have been confirmed together with recently five suggested subtypes 100 . In this study, ST1, ST5, ST7, ST10, and ST14 were reported from samples. Molecular epidemiology studies represented no host-specificity among subtypes, although some subtypes are frequently reported from certain hosts. In this study, ST1 and ST7 were the only human-prevalent subtypes, which were detected from cattle and chicken, respectively. ST1 was allele 4, which is commonly reported among ST1 isolated from humans, as well. This finding may highlight the importance of humans to animals and vice versa besides human to human transmission for this subtype. ST7 is an originally avian subtype, which has been reported from humans, as well [101][102][103] . Our finding showed that one of Blastocystis sp., isolated from chickens was ST7 allele 99. To our best of knowledge, allele 99 was only detected in a recent study by Mohammadpour et al. (2020) 99 who characterized ST7 allele 99 from stool samples of dogs in south of Iran. The presence of avian subtypes such as ST7 among humans suggests the probability of zoonotic transmission from avian source 102,103 .
As result, ST10 was the major subtype identified in sheep and was also detected from cattle and a chicken. ST10 has been frequently reported from sheep and cattle with majority reports from sheep 98,[104][105][106] . Notable, the presence of ST10 in chickens is not a common phenomenon and there is limited data on the report of this subtype in birds 98 . Although pseudoparasitism should be ruled out, cross-transmission of subtypes of Blastocystis sp. between different hosts appears to be a probable event. All ST10 in our study represents allele 152. Data on the allele distribution of ST10 is insufficient. In a most recent study, Mohammadpour et al. (2020) 99 , characterized allele 152 among stool samples from cats and dogs, which support the hypothesis suggesting cross-transmission of ST10 among animals. ST14 is a major subtype reported from sheep and cattle; however, there is no sufficient data on allele distribution of this subtype (Table 7). Table 6. A summary of distribution of the genotypes D, BEB6, I, and horse1 from selected studied hosts (sheep, cattle, chicken, and horse) in the world. *Due to lack of access to supplementary tables, the number of the genotype BEB6 is attributed to all E. bieneusi-positive samples. In studies that worked on several hosts, the number of samples, positive samples, and the genotypes were justified based on the only selected hosts (cattle, sheep, chicken, and horse) and investigated genotypes.

Conclusion
The current study provides interesting data about the prevalence of Blastocystis sp., and E. bieneusi and their subtypes/genotypes among livestock. The presence of zoonotic potential genotypes of E. bieneusi in animals in this study increases the concerns on emerging microsporidia infections among humans who are in close-contact with livestock. Despite of levels of host-adaptation among the genotypes I, BEB6, and horse1 in our study, our findings propose high probability of cross-transmission of E. bieneusi among different hosts. In addition, characterization of zoonotic potential subtypes ST1 and ST7 among our samples provides a clue about the transmission dynamic of Blastocystis sp. between animals-animals and humans-animals, which needs further investigations with larger sample size.   Performing real-time PCR amplification. Four different specific primers targeting ribosomal genes of Cryptosporidium spp., Blastocystis sp., E. bieneusi, and Encephalitozoon spp., were selected (Table 8).
Real-time PCR was carried out using Rotor-Gene Q (QIAGEN, Germany) real-time instrument. The realtime PCR reactions were conducted in a 15-μL total volume containing 7.5 μL of 2 × real-time PCR master mix (BIOFACT, Korea), 0.5 μL of each primer (5 ρM), 3.5 μL of distilled water, and 3 μL of template DNA. Amplification reactions were done as follows: 95 °C for 10 min followed by 40 cycles: 95 °C for 25 s, 59 °C for 30 s, 72 °C for 20 s, and ramping from 70 °C to 95 °C at 1°Cs −1 . Appropriate positive sequenced controls for each parasite together with sterile distillated water as negative controls were tested in each run. The real-time PCR assays were carried out in duplicate to check the reproducibility. The melting profiles were also analyzed using Rotor-Gene Q software to exclude non-specific amplifications and primer-dimers.
Real-time PCR results were considered negative when the Ct value was more than 38 or no amplification curve was obtained. All samples with Ct value above 35 were either retested or their melting curve were justified by the positive control to confirm the result.
Blastocystis subtyping. The barcoding region of Blastocystis sp. was amplified using primers RD5 (5′-ATC TGG TTG ATC CTG CCA GT-3′) and BhRDr (5′-GAG CTT TTT AAC TGC AAC AACG-3′) 130 in samples, which were positive using real-time PCR. Positive sequenced isolates of E. bieneusi and Blastocystis sp. together with sterile distillated water were included in each PCR run as positive and negative controls, respectively. To visualized the targeted fragments, 5 μL of PCR products was electrophoresed on 1.5% of agarose gel in TBE (0.09 M Tris, 0.09 M boric acid, 2 mM EDTA), stained with 0.5 μg/mL ethidium bromide, and visualized with UV transilluminator (Cleaver Scientific Ltd, Warwickshire, United Kingdom). All positive PCR products were sequenced using an ABI 3130 sequencer (Applied Biosystems, USA).
To characterize the genotypes and subtypes of E. bieneusi and Blastocystis sp., respectively, generated sequences were compared in the basic local alignment search tool (BLAST) search (http:// www. ncbi. nlm. nih. gov/ blast/) and then aligned and analyzed together with references orthologs, downloaded from the GenBank database, by the ClustalW program incorporated in BioEdit v. 7.2.6 software. Moreover, to obtain the alleles of Blastocystis sp. subtypes, the sequences of each subtype were subjected to typing tool (http:// pubml st. org/ blast ocyst is/) database. The generated sequences were submitted in the GenBank database with accession numbers MW429392-MW429429 and MW426210-MW426254 for E. bieneusi and Blastocystis sp., respectively. Phylogenetic analysis. Phylogenetic trees were drawn for the ITS fragment and the barcoding region of E. bieneusi and Blastocystis sp., respectively, using the maximum-likelihood algorithm and Tamura-3-parameter substitution model in MEGAX software (http:// www. megas oftwa re. net/) 131 . Bootstrap analyses with 1000 replications were employed to test the reliabilities of the trees. A number of reference sequences retrieved from the GenBank database were also included, alongside with our sequences to evaluate the phylogenetic relationships among isolates. Statistical analysis. Statistical analysis was performed using SPSS version 23 software (SPSS Inc., IBM, Chicago, IL, USA). Pearson's chi-squared (χ 2 ) for independence and Fisher's exact two-sided tests were conducted to evaluate the prevalence and association between parasite and animals. A P value < 0.05 was considered statistically significant.