Blastocystis has been reported in humans worldwide. The prevalence of Blastocystis usually varies based on geographical regions and studied population [31, 32]. The prevalence of Blastocystis has been reported in humans ranged from 0.5% in Thailand to 100% in Senegal [6]. However, in a study, Blastocystis as a common member of the healthy human gut microbiota is present in 56% of sample-sets which is much higher than previously reported from an industrialized country (Ireland) [33, 34]. In the present study, the overall prevalence of Blastocystis in healthy humans reached 14.0% (Table 1), which corresponds well with prevalence data from a previous study ranged from 0.007–43.3% in China [25]. The differences in prevalence may be related to the following factors such as age, contacting animals, traveling, diet, hygienic standard or drinking habits [3]. In the present study, the prevalence was observed in humans in contact with domestic animals, which were considered statistically significant compared to those who did not have contact with animals (Table 2). This result was consistent with previous studies, and suggested that humans were close contact with livestock or birds could increase the risk of Blastocystis infection [3, 35]. The prevalence of Blastocystis varies in mammals between and within countries worldwide and is as high as 100% in some studies, such as in pigs from China and cattle from Indonesia [27, 36]. Nevertheless, Wang et al. (2018) described the prevalence as low as 9.5% in cattle, 8.8% in pigs, with an absence in goats in China’s Heilongjiang Province [37]. In the present study, Blastocystis sp. was detected in three mammal species (pigs, goats and cattle) and one bird species (chickens), with the prevalence in domestic animals ranged from 2.1% in chickens to 71.8% in pigs. Goats infected with Blastocyst sp. were reported for the first time of Heilongjiang Province and the prevalence was 50% (7/14) (Table 1). However, domestic animals infection with Blastocystis sp. were present in 17.9% (46/257) of our sample set compared to other studies where prevalence ranged from 6.3–81.4% [4, 7, 38–40]. The fact of low prevalence could be explained by that a large proportion of domestic animal samples were from poultry, in which an absence of Blastocystis were found in geese and ducks and Blastocystis was only found in three chickens. Similar to our finding, Blastocystis sp. infection was absence in examined geese and ducks in UK [5], in Côte d’ Ivoire [41], in Malaysia [38], in Algeria [42], in Italy [43], in Mauritius [5]. Additionally, the stool samples from free-roaming poultry were collected directly from the ground in November and in February in Northeast China. The low temperature and the lower stocking density may be decrease the chance of transmission of Blastocystis sp. in poultry [44, 45]. Furthermore, most of the poultry were vaccinated. Previous studies showed that vaccine may protect against bacterial, fungal, parasitic or viral infections in animals [46].
Molecular epidemiological studies have revealed a significant difference in the distribution of subtypes across host species and geographical regions [31]. ST3 is the most common subtype in humans. The second most common is ST1 in humans except the studies from continental Europe and the UK, where ST4 is common [6]. In China, ST3 and ST1 in humans are common subtypes in eight provinces, excluding the study from Hebei province, where ST2 is predominant [28]. In the present study, the most common subtype in the human sample set was ST3 (62.5%; 5/8), followed by ST1 (25%; 2/8) and ST2 (12.5%; 1/8) (Table 1). Our result was consistent with most studies, although the difference in constitutions and ratios of Blastocystis subtypes might vary in different populations studied [3, 47]. Balstocystis ST2 (allele 9) was only detected in an elderly man from YH and this is the first report of Blastocystis ST2 in humans in Heilongjiang Province. However, ST2 as common subtype was found in children of China’ s Hebei Province and Senegalese [28, 48]. According to a previous study, ST2 was commonly identified in fecal samples of cynomolgus monkeys [49], and was also identified in domestic animals [11]. Humans were in frequent contact with monkeys and domestic animals will increase the risk of infection with Blastocystis [11, 50]. In the present study, because of the absence of Blastocystis ST2 in domestic animals in the investigated areas, we cannot give a satisfactory explanation for zoonotic transmission of Blastocystis.
Interestingly, the present study allowed us to see if Blastocystis sp. and its subtypes could circulate within family members or domestic animals. We obtained samples from 33 families and detected at least one Blastocystis sp. subtype in 13 families (Table 3). However, in one DFH family that raised pigs, we discovered two members infection with Blastocystis ST1 (a4). The result might suggest that Blastocystis sp. circulates between two members or they could have been colonized from the same source [3] (Fig. 1). If humans are asymptomatic or they have poor personal hygiene within a family, infected person can easily spread the parasite to others [7]. Meanwhile, in the present study, ST1 (a4) was identified not only in two humans in one family, but also in one pig from another family in DFH. The Blastocystis ST1 sequences of pig (P20) and two humans (H36, H37) were clustered into one branch with a high bootstrap value, which may further be suggested potential transmission of Blastocystis infection between humans and pigs in DFH (Fig. 2). Blastocystis ST1 are considered be zoonotic and observed in some other animals, such as livestock, companion animals, birds, non-human primates, wildlife [11]. In the present study, besides ST1, ST5 are the most predominant subtypes in pigs in DFH and YH. Because of a low prevalence of Blastocystis ST1 (7.1% 1/14) in DFH pigs (Table 1), it seems unlikely that pigs are a zoonotic source of Blastocystis ST1 infection in this area. Our results are consistent with result of previous study in pigs in Thailand [7]. Therefore, the role of pigs to transmit Blastocystis ST1 should be further evaluated in the future studies.
As for Blastocystis ST3, which has been classified as more anthroponotic subtype and was also reported from animals closely contacted with humans, such as cattle, pigs, sheep, racoon dogs, chickens, non-human primates, dogs and cats [11, 51]. In the present study, Blastocystis-infected three humans and one cattle with the same ST3 (a34) were found from different families in DFH (Table 3). The result can likely be explained by reverse zoonosis from human handlers to animals, as suggested in previous studies focused on various animal hosts (pigs, cattle, cows, sheep, goats, dogs, cats, chickens and ducks) [11, 52]. In the present study, apart from ST3, ST10 and ST14 were predominant in cattle (Table 1). Blastocystis ST10 and ST14 were also observed in other hosts, such as ST10 was observed in dogs, cats, pigs, sheep, goats, rodents, ostriches, monkeys, wild and zoo hoofed animals and ST14 was found in sheep, goats, rabbits, birds, wild cat, wild and zoo hoofed animals [11]. Meanwhile, some studies have reported that cattle may be assigned as specific host for ST10 and ST14, which globally represented the most widely distributed subtypes, such as in Korea, Lebanon, Turkey, China and United States [11, 13, 15, 53–55]. However, in a recent study, ST10 and ST14 have also been identified in Senegalese school children, who were infected with Blastocystis sp. may be through either direct contact with livestock or water-borne transmission [48]. In the present study, ST10 and ST14 were predominant in cattle, followed by ST3 (Table 1). Thus, Blastocystis ST10 and ST14 in cattle may play a prominent role in the transmission of zoonotic Blastocystis subtypes to humans, which should be considered for implementation of future preventive measures [15].
According to the results of previous studies, pigs are natural host for Blastocystis ST5, due to most of Blastocystis-positive pigs harbored ST5 regardless of geographical setting [7, 20, 56]. Besides pigs, ST5 has also been detected in some domestic animals (cattle, sheep, goats) and wildlife (blesbok, oryx, grey brocket, wild boar, black rhinoceros, wild cat) [11] Although ST5 is considered rare in humans, it has been reported to be a potential zoonosis in rural China [27]. In the present study, Blastocystis ST5 (a153) was not only identified in one pig, but also in two goats in one family. While, Blastocystis ST5 (a115) was also found in three pigs and one goat from different families in DFH. Blastocystis ST5 was not detected in any human samples from the studied areas. All ST5 sequences had 99–100% similarity with ST5 isolated from animal-derived sequences in GenBank (Table 5). It suggested that this parasite could be transmitted from pigs to goats or goats to pigs in DFH [7]. In addition, fecal contamination of grass and fodder was common, which may increase the risk of Blastocystis infection in animals [10]. Interestingly, a118 (ST5) in pig is a novel finding: there are no reports of the allele in pig globally.
Table 5
Homology analysis of nucleotide sequences of Blastocystis subtypes
Host | Isolate ID | ST/allele (n) | GenBank ID (host) b | Homology (%) |
Human | DFH (H36, H37) | ST1/a4 (n = 2) | EU445486 (Pig) | 100 |
| DFH (H6, H39, H40) | ST3/a34 (n = 3) | MN338083 (Monkey); MK934333 (Human) MW888497 (Racoon dog); AB107963 (Pig); AB107965 (Cattle) | 100 |
| YH (H44) | ST2/a9 (n = 1) | MN326606 (Human); OL623671 (Human); MN836828 (Human) | 100 |
| SF (H52, H54)a | ST3/a122 (n = 2) | MN339604 (Dog); MK782518 (Human) | 99.65 |
Pig | DFH (P20) | ST1/a4 (n = 1) | EU445486 (Pig); MN585811 (Human); MK782495 (Human) | 99.83 |
| DFH (P3, P14, P21); YH (P38) | ST5/a115 (n = 4) | MK801386 (Pig); MN493729 (Wild boar); MF991106 (Sheep) | 100 |
| DFH (P5, P12, P15, P17, P19, P22, P23, P24, P27); YH (P29, P30, P31, P33, P36) | ST5/a153 (n = 14) | MK801419 (Pig); MF186709 (Wild boar); MW850525 (Sheep) | 100 |
| DFH (P28) | ST5/a118 (n = 1) | MW242641 (Squirrel); KY989561 (Pig) | 100 |
Cattle | DFH (B2, B3) a | ST14/a157 (n = 2) | MW426241 (Cattle) | 100 |
| DFH (B4) | ST3/a34 (n = 1) | MN338083 (Monkey); MK934333 (Human) MW888497 (Racoon dog); AB107963 (Pig); AB107965 (Cattle) | 100 |
| DFH (B5, B6) | ST10/a43 (n = 2) | MF1866689 (Deer) | 100 |
Goat | DFH (S1) | ST5/a115 (n = 1) | MK801421 (Pig); MK085081 (Cattle); MN493729 (Wild boar); MW888498 (Racoon dog); MF991106 (Sheep) | 100 |
| DFH (S2, S3) | ST5/a153 (n = 2) | MK801419 (Pig); MF186709 (Wild boar); MW850525 (Sheep) | 100 |
Chicken | YH (C137) | ST7/a96 (n = 1) | MK010975(Chicken); MW867033 (Quail); FJ809939 (Human) | 100 |
| SF (C84)a | ST7/a99 (n = 1) | MF326200 (Chicken) | 100 |
a: allele. |
a Closest match: 18S rRNA full length: a122; a157; a99. |
b Accession no. indicating the sequences downloaded from GenBank, which have 99–100% homology with the sequences obtained in the present study. |
ST6 and ST7 were considered “avian STs’’, because of their relative predominance in birds [1]. In Brazil, Poland, Czech Republic and Lebanon, there were several reports of ST6 or ST7 in humans [1, 3, 23]. Indeed, ST6 isolates were found to transmit from chickens to two staff members of a poultry slaughter house in Lebanon, probably because of repeated exposure to a large amount of chicken feces [1]. Moreover, in Brazil, ST7 was detected from two children and a household member, as well as an asymptomatic individual and a transplant candidate [23, 57, 58]. In the present investigated areas, only ST7 (a99), ST7 (a96) were found in chickens in SF and YH, respectively. However, one Balstocystis ST7 (a96) sequence from chicken in YH was identical to that from human (FJ809939) in China, suggesting potential zoonotic transmission of Blastocystis infection (Table 5).
Based on the currently known subtypes, all subtypes were clearly separated by the phylogenetic analysis. However, Blastocystis ST3 sequences originating from DFH and SF humans formed two distinct clades (Fig. 2). Therefore, the distribution of Blastocystis ST3 is clearly subject to a variation in the geographic locations [32]. In pairwise distance comparisons, between ST3 were found to have large variations (Table 4). Additionally, the fact that ST3 (a34) was identical in humans and animal (cattle) in DFH, which was differed from ST3 (a122) found in humans in SF. This result showed the possible potential for between humans and animals transmission of Blastocystis subtype in the same area.