Background

Giardia duodenalis and Enterocytozoon bieneusi are common human pathogens. At present, there are more than 200 million of annual giardiasis cases in humans, while microsporidiosis is a common cause of diarrhea [4,5,6]. Over 200 giardiasis outbreaks have been reported in the world during the period 2004–2016, while E. bieneusi also caused an outbreak in France [6,7,8,9].

Non-human primates (NHPs) are important experimental animals in public health research because of their high genetic similarity to humans [10]. A growing number of studies have found that NHPs are the hosts of many parasites, including gastrointestinal protists E. bieneusi and G. duodenalis, which are transmitted in similar fecal-oral routes [20,21,22]. Thus, genoty** E. bieneusi in NHPs can help us understand the zoonotic potential of E. bieneusi in these animals.

At present, more than 50 E. bieneusi genotypes have been found in NHPs, most of which belong to Group 1 [23]. Among them, genotypes A, D, Type IV, EbpC, Peru7, Peru8, Peru11, PigEBITS7, Henan-V, WL15, I and BEB6 have been found in humans in several countries, including China [6, 6, 31]. Therefore, NHPs are potential reservoir hosts for zoonotic transmission of E. bieneusi.

Similarly, eight distinct G. duodenalis assemblages (A-H) have been identified by genetic analysis of triosephosphate isomerase (tpi), ssrRNA, β-giardin (bg), glutamate dehydrogenase (gdh) and other genes [39, 40]. Multilocus genoty** (MLG) has been used in several studies to understand the host specificity and zoonotic potential of assemblage B in human and NHPs [41,42,43,44]. Controversies exist on the differences in virulence between assemblages A and B in humans [51]. A maximum likelihood (ML) tree was constructed in MEGA v.6 (https://www.megasoftware.net) using evolutionary distances calculated by the commonly used general time reversible model. The reliability of clusters formed was assessed by bootstrap analysis using 1000 replicates.

Statistical analysis

Differences in E. bieneusi and G. duodenalis detection rates between groups of different sex, age, or fecal consistency were assessed by using the Chi-square test implemented in SPSS Statistics v.20.0 (IBM Corp., Armonk, NY, USA). The difference was considered significant when P < 0.05.

Results

Occurrence of E. bieneusi and G. duodenalis in crab-eating macaques

Of the 1452 specimens analyzed, 461 (31.7%) were positive for E. bieneusi. Significantly higher detection rates of E. bieneusi were identified in animals with loose stools (41.4%, 151/365) than animals with normal stools (28.5%, 310/1087; χ2 = 20.83, P < 0.0001), in males (36.5%, 258/706) than females (26.7%, 160/599; χ2 = 14.391, P = 0.0001), and in old animals (> 3 years; 38.6%, 135/350) than young animals (1–3 years; 29.6%, 326/1102; χ2 = 9.90, P = 0.0016; Table 1).

Table 1 Distribution of Giardia duodenalis and Enterocytozoon bieneusi genotypes in crab-eating macaques in Hainan, China by fecal consistency, sex and age
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For G. duodenalis, 362 (24.9%) specimens were positive by tpi PCR, 315 (21.7%) by bg PCR and 240 (16.5%) by gdh PCR. Altogether, 469 (32.3%) specimens were positive for G. duodenalis in at least one PCR. Significantly higher detection rates of G. duodenalis were found in animals with loose stools (41.1%, 150/365) than animals with normal stools (29.3%, 319/1087; χ2 = 17.25, P < 0.0001), and in 1–3 year-old monkeys (36.6%, 403/1102) than older animals (18.9%, 66/350; χ2 = 38.11, P < 0.0001). Nevertheless, detection rates of G. duodenalis were comparable between males (33.4%, 236/706) and females (31.2%, 233/746; Table 1).

Distribution of E. bieneusi genotypes

Nine E. bieneusi genotypes were obtained from PCR-positive specimens by sequence analysis, namely Type IV (236/461), CM1 (119/461), Peru8 (42/461), Pongo2 (27/461), CM2 (17/461), Peru11 (12/461), D (4/461), CM3 (3/461) and PigEbITS7 (1/461).

Among them, eight E. bieneusi genotypes were found in animals with loose stools, namely Type IV (74/151), CM1 (40/151), Pongo2 (12/151), Peru8 (11/151), CM2 (8/151), Peru11 (4/151), PigEbITS7 (1/151) and D (1/151). Similarly, eight E. bieneusi genotypes were detected in animals with normal stools, namely Type IV (162/310), CM1 (79/310), Peru8 (31/310), Pongo2 (15/310), CM2 (9/310), Peru11 (8/310), CM3 (3/310) and D (3/310). A similar distribution of E. bieneusi genotypes was also seen between male and female monkeys as well as young and old monkeys (Table 1).

Distribution of G. duodenalis genotypes and subtypes

Sequence analysis of PCR products from the tpi, bg and gdh genes showed that all 469 G. duodenalis-positive specimens had assemblage B (Table 1). Eight G. duodenalis subtypes were obtained from the 362 PCR-positive specimens at the tpi locus, including four known and four new subtypes. Among them, B-sh01 (n = 108), B1 (n = 75), B6 (n = 27) and B2 (n = 17) found in this study were identical to reference sequences JX994245, KC441076, GU564284 and KC441077, respectively. The new subtypes B-hn02 (n = 78), B-hn04 (n = 32), B-hn01 (n = 13) and B-hn03 (n = 12) had one, one, two and one single nucleotide polymorphism (SNP), respectively, compared with the reference sequence MF095053 (Table 2).

Table 2 Intra-genotypic nucleotide substitutions in the triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh) and β-giardin (bg) genes of Giardia duodenalis in crab-eating macaques in Hainan, China
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Seven G. duodenalis subtypes were present among the 315 PCR-positive specimens at the bg locus, including four known and three new subtypes. Among them, B-CD10 (n = 171), B2 (n = 59), B-Egyh8 (n = 58) and B-VANC/91/UBC/67 (n = 5) found in this study were identical to reference sequences KY696837, MG736242, KC441079 and KM190799, respectively. The new subtypes B-hn08 (n = 20), B-hn06 (n = 1) and B-hn07 (n = 1) had four, two, and one SNP, respectively, compared with the reference sequence KY696837 (Table 2).

Seven subtypes of G. duodenalis assemblage B were detected among the 240 PCR-positive specimens at the gdh locus, including six known ones and one new subtype. Among them, B-VANC/96/UBC/127 (n = 162), B-VANC/87/UBC/8 (n = 40), B-VANC/91/UBC/67 (n = 9), BIV (n = 7), B-Afu97 (n = 5) and B-sh03 (n = 2) found in this study were identical to the reference sequences KM190707, KM190714, KM190708, KF679733, HM134210 and JX994233, respectively. The new subtype B-hn05 (n = 15) had three SNPs compared with the reference sequence KM190707 (Table 2).

Multilocus genoty** of assemblage B

Of the 469 specimens positive for G. duodenalis assemblage B, 161 were positive by PCR at all three genetic loci. They belonged to 53 MLGs (MLG-B-hn01 to MLG-B-hn53). Among them, MLG-B-hn01 (16.7%) was the most common, followed by MLG-B-hn02, MLG-B-hn03 and MLG-B-hn04, with frequencies of 7.5%, 6.2%, and 5.0%, respectively. In contrast, the frequency of MLG-B-hn05 and MLG-B-hn06 was 4.3%, the frequency of MLG-B-hn07 and MLG-B-hn08 was 3.7%, while the remaining MLGs were each seen in fewer than five specimens (Table 3).

Table 3 Multilocus sequence genotypes of Giardia duodenalis assemblage B in crab-eating macaques in Hainan, China
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Phylogenetic relationship of G. duodenalis assemblage B

Phylogenetic analysis of concatenated sequences of the 53 assemblage B MLGs in this study, and those from previous studies [51] showed that most MLGs from this study were related to MLGs previously found in Old World monkeys (MLG-3, MLG-4, MLG-7, MLG-8, MLG-14 and MLG-15). However, one of the MLGs, MLG-B-hn31, seen in one animal, clustered together with MLGs in humans. In addition, MLG-B-hn42 and MLG-B-hn43 were genetically separated from Old World monkeys, ring-tailed lemurs and humans (Fig. 1).

Fig. 1
figure 1

Phylogenetic relationship of multilocus genotypes (MLGs) of Giardia duodenalis assemblage B inferred by the maximum likelihood analysis of concatenated tpi, gdh and bg nucleotide sequences using genetic distances calculated by the general time reversible model (GTR). Reference sequences (MLG1-15, isolates Sweh001, Sweh059, Sweh074, Sweh107, Sweh136, Sweh158, ECUST1710, ECUST5414, ECUST4064 and ECUST981) used are from the studies by Lebbad et al. [51], Karim et al. [18] and Wang et al. [27]. Bootstrap values greater than 50% from 1000 replicates are shown on nodes. MLGs identified in the present study are in bold. The scale-bar indicates 50 nucleotide substitutions per 100 nucleotides

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Discussion

Data from this study suggests that crab-eating macaques in Hainan, China are commonly infected with E. bieneusi. In this study, the detection rate of E. bieneusi in these animals was 31.7% (461/1452). This is higher than the reported detection rates in NHPs in various countries [52,53,54,55]. Similarly, it is mostly higher than detection rates in studies of E. bieneusi in NHPs in China [18, 19, 35, 59]. Many of the studies reporting low detection rates of E. bieneusi in NHPs were performed using wild, captive and zoo animals [19, 35, 52, 54, 55, 63, 64] and different areas within China [34, 35, 38, 44, 65]. The very high detection rate of G. duodenalis as well as E. bieneusi in the present study could be attributed to the intensive farming of NHPs in this study, which congregates numerous susceptible individuals in confined areas.

To date, assemblages A, B and E of G. duodenalis have been reported in NHPs [34, 38, 65,66,67]. Among them, assemblage B is the most common genotype in different species of NHPs, including various monkeys, lemurs, gibbons, chimpanzees and gorillas [34, 36,37,38, 63,64,65]. It is also common in humans in both develo** and industrialized countries, and is more common than the other major human-pathogenic genotype, assemblage A [51]. In contrast, most of other MLGs were genetically related to assemblage B isolates in pig-tailed macaques, rhesus macaques, golden monkeys, yellow baboons and green monkeys, all common Old-World monkeys. They were different from MLGs in ring-tailed lemurs, which are natives of the island nation Madagascar and evolved independently from monkeys and apes.

Conclusions

In this study, we have shown a frequent occurrence and high genetic diversity E. bieneusi and G. duodenalis subtypes in crab-eating macaques in one commercial laboratory animal facility in Hainan, China. Most of the E. bieneusi genotypes and G. duodenalis assemblage B subtypes are potentially zoonotic. Additional genetic characterizations of these pathogens at other genetic loci, including more conservative ones for G. duodenalis, are needed to better understand the transmission of these pathogens and possible occurrence of host segregation within G. duodenalis assemblage B. Measures should be implemented at the commercial facility to reduce the transmission of enteric parasites.